Porphyrin Compounds and Compositions Useful for Treating Cancer

ABSTRACT

A porphyrin compound of Formula IIIand composition made therefrom comprising a therapeutically effective dose of a porphyrin bound via a linker to an anti-cancer agent useful in treating cancer in a patient in need thereof or to treat cancer cells in-vitro. The compounds and compositions may be delivered by a drug delivery device as disclosed here and be part of a kit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/222,661, titled “Porphyrin Compounds and Compositions Useful forTreating Cancer”, filed on Dec. 17, 2018, which is a continuation ofInternational Patent Application No. PCT/US2017/037982, titled“Porphyrin Compounds and Compositions Useful for Treating Cancer”, filedJun. 16, 2017, which claims priority to and the benefit of the filing ofU.S. Provisional Patent Application No. 62/351,165 titled “Compositionfor Treating Cancer and Method of Use”, filed on Jun. 16, 2016, and thespecification and claims thereof are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable.

COPYRIGHTED MATERIAL

Not Applicable.

BACKGROUND OF THE INVENTION

Macrocyclic structures having multiple pyrrole rings joined in amacrocycle are dynamic molecules involved in many biological processes,including oxygen and electron transport. Porphyrins are natural pigmentscomprised of four pyrrole rings connected via four methine (═CH—)carbons to form an aromatic macrocycle. The IUPAC system is used innumbering positions in a porphyrin. There are a total of 24 positions inthe porphyrin ring, including the nitrogen atoms. Carbon atoms areassigned numbers 1-20, starting at the α position, and going around theperiphery of the entire heterocycle. α positions are assigned numbers 1,4, 6, 9, 11, 14, 16 and 19, whereas β positions are assigned numbers 2,3, 7, 8, 12, 13, 17 and 18. The meso positions (carbon atoms at themethine bridges) are numbered 5, 10, 15, and 20. The nitrogen atoms onthe other hand are numbered 21, 22, 23 and 24.

Some porphyrins are isolated from natural sources, for example,protoporphyrin IX is the organic portion of iron-containing porphyrinhemin. Many other porphyrins are prepared synthetically. These includethose made via the condensation of aldehydes and pyrroles, such astetraphenylporphyrin, made from the condensation of benzaldehyde andpyrrole. Some porphyrins are useful in photodynamic therapy whenactivated at an excitation wavelength (for example, 415 nm) for thetreatment of cancer. Some cationic porphyrins demonstrate non-covalentinteractions with DNA. Aside from interacting strongly with DNA,cationic porphyrins can also cleave DNA, have high photonuclease andphotodynamic therapy (PDT) application, and have been found to inhibittelomerase through G-quadruplex stabilization and or to inhibittranslation via binding to G-quadruplex tetra-meso (N-methyl-4-pyridyl)porphine and C14-alkyl derivative tri-meso(N-methyl-4-pyridyl),meso(N-tetradecyl-4-pyridyl) porphine (called C14). Other porphyrinshave been shown to be activated by ultrasound such as the compoundsdescribed in Cancer Sci. June 2007, vol. 98; no. 6 pgs. 916-920.

Porphyrins have been shown to be taken up by cancer cells preferentiallyover normal cells in tissue culture experiments, animal models as wellas human patients. This cancer cell selectivity is in part responsiblefor the utility of porphyrins in photodynamic therapy. The mechanism ofporphyrin localization in tumors is not well understood. Understandingthis mechanism could have important implications for improved selectivedelivery of porphyrin compounds to tumors and the targeted delivery ofcytotoxic drugs to tumors.

A variety of cancer therapies and treatments exist such as surgicalresection of solid tumors, radiation, and chemotherapy. While surgicalresection and radiation is used on localized tumors, chemotherapy isoften delivered systemically and impacts both cancer and non-cancercells because the traditional chemotherapy enters both cell types; thereis no preference for entering cancer cells vs. non-cancer cells. Becauseof this, chemotherapy is often associated with unwanted toxicity, whichcan even lead to death. A cancer treatment using a compound orcomposition that preferentially enters a cancer cell as compared to anon-cancer cell is therefore desired.

Porphyrins show higher binding to and/or internalization in cancer cellsas compared to non-cancer cells. The mechanism responsible for this ispoorly understood. Literature data suggests that the endocytoticpathways may be a mechanism for preferential porphyrin internalizationby cancer cells. In addition, previous studies have suggested that someporphyrins interact with the low-density lipoprotein receptor (LDLR) topreferentially enter cancer cells vs. non-cancer cells. Based upon thisinformation, designing porphyrin compounds that take advantage of anLDLR interaction (or other to be identified endocytosis-relatedreceptors) could improve the activity of porphyrin compounds used totreat cancer cells.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

One embodiment of the present invention comprises a compound of formulaIII

-   -   or a salt thereof, wherein    -   an A₁, A2, A3 and A4 are each covalently attached to a porphyrin        ring and A₁, A2, A3, and A4 are independently selected from a        substituted aromatic ring or a six-membered heteroaromatic ring        containing a single nitrogen atom at the 2, 3 or 4 position        relative to the porphyrin ring;    -   B₁ is selected from the group consisting of L9-L16 wherein n is        selected from 1-12; and    -   Z₁ is a cytotoxic agent selected from the group consisting of        T1b, T2b, T3b, T4b, 1, T1a, T3a, T4a, T8a, T10a, T14a, T15a,        T18a, T19a, T21a, T27a, T31a, T32a, T33a, T4c, T5c, T9c, and        T10c and derivatives thereof.

The substituted aromatic ring of the A₁ of the Formula III compound or asalt thereof may comprise a carboxylic amide functional group at eitheran ortho, meta, or para position with respect to the porphyrin ring andwherein A2, A3 and A4 are each a substituted aromatic ring wherein eachA2, A3, and A4 substituted aromatic ring has a substituent at either aortho, meta or para position with respect to the porphyrin ring and thesubstituent is either a carboxylic acid or carboxylic methyl ester. Forexample, the substituted aromatic ring of the A2, A3, and A4 of theFormula III compound or a salt thereof comprises a carboxylic methylester in a para position with respect to the porphyrin ring and thecarboxylic amide of A₁ is in the para position with respect to theporphyrin ring.

In one embodiment of the present invention, the B₁ of the Formula IIIcompound or a salt thereof is L11 or L13.

In one embodiment of the present invention, the Z₁ of the Formula IIIcompound or a salt thereof is selected from the group consisting of T1b,1 and T4c.

In another embodiment of the present invention, the substituted aromaticring of the A₁ Formula III compound or salt thereof comprises anaromatic ether functional group at either an ortho, meta or paraposition with respect to the porphyrin ring, and wherein A2, A3 and A4are each the substituted aromatic ring wherein each A2, A3, and A4substituted aromatic ring has a substituent located at an ortho, meta orpara position with respect to the porphyrin ring wherein the substituenton each A2, A3, and A4 substituted aromatic ring is independentlyselected from the group consisting of: lower alkyl, branched loweralkyl, cycloalkyl, halogens (F, Cl, Br, I), cyano, amino or substitutedamino, sulfonic acid or sulfonamide, aromatic ether, aromatic hydroxyl,carboxylic acid alkyl esters or carboxylic acid amide.

In one embodiment of the present invention, the B₁ of the Formula IIIcompound or a salt thereof is selected from the group consisting of: L9,L10, L15, and L16.

In one embodiment of the present invention, a substituent of thesubstituted aromatic ring at position A2, A3 and A4 of the Formula IIIcompound or a salt thereof is a hydroxyl and may occupy the ortho, metaor para position with respect to the porphyrin ring and B₁ is L9 or L15.

In one embodiment of the present invention, the substituted aromaticring A₁ of the Formula III compound or a salt thereof comprises anaromatic ether functional group, where the position of the aromaticether is meta with respect to the porphyrin ring, and wherein A2, A3 andA4 are each the substituted aromatic ring wherein the substituent on thesubstituted aromatic ring is an aromatic hydroxyl in the meta positionwith respect to the porphyrin ring, B₁ is L9 or L15 and Z₁ is selectedfrom the group consisting of: T1b, 1 and T4c.

In one embodiment of the present invention, the six-memberedheteroaromatic ring of A₁ of the Formula III compound or a salt thereofcomprises a nitrogen atom where a position of the nitrogen atom on thesix-membered heteroaromatic ring may occupy one of a 2, 3 or 4 positionwith respect to the porphyrin ring, A2, A3 and A4 are each a pyridinering where the position of a pyridine nitrogen on each pyridine ring ofA2, A3 and A4 may independently occupy one of the 2, 3 or 4 positionwith respect to the porphyrin ring. Further still B₁ is selected fromthe group consisting of: L9, L10, L15, and L16. In one example, thesix-membered heteroaromatic ring comprising the nitrogen atom at A₁ is apyridinium where the position of the nitrogen is in the 4 position withrespect to the porphyrin ring, B₁ is L9 or L15, and Z₁ is selected fromthe group consisting of T1b, 1 or T4c. Further still, B₁ is L9.

In one embodiment of the present invention the Formula III compound or asalt thereof is selected from the group consisting of: OS002, OS007,OS009, OS0030, OS0032 and OS0035.

In one embodiment of the present invention the Formula III compound or asalt thereof is selected from the group consisting of: OS0023 andOS0024.

In one embodiment of the present invention the Formula III compound or asalt thereof is selected from the group consisting of: OS0025, OS0026,OS0027, and OS0029.

Another embodiment of the present invention provides for a method oftreating cancer in a patient in need thereof comprising the steps of:administering to a patient in need thereof a therapeutically effectiveamount of the Formula III compound or a pharmaceutically acceptable saltthereof.

Another embodiment of the present invention provides for a method oftreating cancer cells in vitro comprising the steps of: administering tothe cancer cells a therapeutically effective amount of the Formula IIIcompound or a pharmaceutically acceptable salt thereof to inducecytotoxicity activity in the cancer cells preferentially as compared tonon-cancer cells.

One embodiment provides for any of the Formula III compound describedherein or a salt thereof, further including a pharmaceutical acceptablecarrier, for example, the pharmaceutically acceptable carrier is aliquid carrier selected from the group consisting of saline, glucose,alcohols, glycols, esters, amides, and any combination thereof.

Another embodiment provides that a Formula III compound as describedherein or a salt thereof is in a dosage form and the dosage form isparenteral and the dosage form is selected from the group consisting ofintradermal dosage form, a subcutaneous dosage form, an intramusculardosage form, a subcutaneous dosage form, an intravenous dosage form, anintrathecal dosage form, and an epidural dosage form. Alternatively, thedosage form is nonparenteral and the dosage form is selected from thegroup consisting of oral dosage form, sublingual dosage form, topicaldosage form, transdermal dosage form, ophthalmic dosage form, oticdosage form, nasal dosage form, rectal dosage form, and vaginal dosageform.

In one embodiment, the salt of the Formula III compound is apharmaceutically acceptable salt.

One embodiment of the Formula III compound or a pharmaceuticallyacceptable salt thereof provides is useful in the treatment of cancer.

Another embodiment of the Formula III compound of or a pharmaceuticallyacceptable salt thereof is useful in the treatment of cancer cells invitro.

In one embodiment, a composition comprises a Formula III compound and acytotoxic agent wherein the cytotoxic agent is a formula that is thesame or different than Z₁ of the compound. For example, the differentformula of the cytotoxic agent is selected from a class that isdifferent as compared to Z₁ the compound.

Another embodiment of the present invention provides for a drug deliverydevice comprising the Formula III compound enmeshed with a biodegradablepolymer. For example, the biodegradable polymer of the drug deliverydevice is selected from the group consisting of poly lactic co-glycolicacid, alginate, and polycaprolactone. Further still, the Formula IIIcompound is released over time when the drug delivery device isimplanted into a patient.

Another embodiment provides for a kit comprising a Formula III compoundor a pharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable carriers.

Another embodiment of the present invention provides for a method oftreating cancer in a patient in need thereof comprising the steps of:administering to a patient in need thereof a therapeutically effectiveamount of the Formula III compound or a pharmaceutically acceptable saltthereof.

One embodiment of the present invention provides a therapeutic compoundcomprising a therapeutically effective dose of a compound comprised of aporphyrin bound via a linker to an anti-cancer agent, sometimes referredto herein as a porphyrin anticancer conjugate (“PAC”) compound. Theanti-cancer agent may be selected from the group consisting of cytotoxicagent, and/or radionuclide also known as radioactive nuclide,radioisotope or radioactive isotope. For example, the anti-cancer agentmay be alkylating agents, antimetabolites, anti-tumor antibiotics,antimicrotubule agents, kinase inhibitors, hormonal agents, monoclonalantibodies, glucocorticoids, mitotic inhibitors, topoisomerase Iinhibitors, topoisomerase II inhibitors, immunomodulating agents,cellular growth factors, cytokines, histone deacetylase inhibitors, andnonsteroidal anti-estrogenic agents but not limited thereto. Atherapeutic composition may further include a PAC compound and apharmaceutical carrier. In one embodiment, the carrier may be anexogenous protein. In another embodiment, the carrier is not anexogenous protein.

Another embodiment provides for a method of treating cancer in a patientin need thereof comprising the steps of administering to a patient inneed thereof a therapeutically effective PAC compound or composition asdisclosed herein.

One aspect of the present invention provides for a method of providingtwo means of cell killing simultaneously via a single compound. A PACcompound will enter the cancer cell, wherein, upon exposure of thecancer cell to laser light of proper emission to excite the porphyrin,the porphyrin irreversibly damages the DNA and the anti-cancer agentacts as a cytotoxic agent in addition.

Another aspect of the present invention provides for a compoundcomprising a plurality of anti-cancer agents, which in combination havea synergistic therapeutic effect on a cancer cell and or patient in needof anti-cancer treatment.

Another aspect of a PAC compound as disclosed herein provides for areduced side effect of the anti-cancer agent alone while maintaining theanti-cancer effects of the agent on the cancer cell or patient in needof anti-cancer treatment.

Another aspect of the present invention provides for a lower toxicity ofthe PAC compound as a treatment agent as compared to a cytotoxic agentadministered individually.

In one embodiment of the present invention, the anti-cancer agent of thePAC compound does not include the following: a polyamine, polyamineanalog, cyclic polyamine, cyclic polyamine analog, dioxonaphthoquinone,or dioxonaphthoquinone antitumor antibiotics, bleomycin, dactinomycin,mitoxantrone, mitomycin, epipodophyllotoxins, etoposide, teniposide,antimicrotubule agents, vinblastine, vincristine, vindesine,vinorelbine, other vinca alkaloids, taxanes, paclitaxel (taxol),docetaxel (taxotere), nitrogen mustards, chlorambucil, cyclophosphamide,estramustine, ifosfamide, mechlorethamine, melphalan, aziridines,thiotepa, alkyl sulfonates, busulfan, nitrosoureas, carmustine,lomustine, and streptozocin, platinum complexes, carboplatin cisplatin,alkylators, altretamine, dacarbazine, procarbazine, temozolamide, folateanalogs, methotrexate, purine analogs, fludarabine, mercaptopurine,thioguanine, adenosine analogs, cladribine, pentostatin, pyrimidineanalogs, capecitabine, cytarabine, floxuridine, fluorouracil,gemcitabine, substituted ureas, hydroxyurea, camptothecin analogs,irinotecan and topotecan, topoisomerase I inhibitors, topoisomerase IIinhibitors, and quinone compounds.

Another aspect of an embodiment the present invention provides for a PACcompound that is taken up by cancer cells such that porphyrinfluorescence as measured from the cancer cells is greater by a factor of2 or more as compared to non-cancer cells when both are exposed to awavelength of light that excites the porphyrin and or sound wave thatexcites the porphyrin.

Another aspect of the present invention provides for treatment of asubject with a PAC compound having the formula Pn-Ln-Tn wherein Pn is aporphyrin, and wherein n is selected from 1-4, Ln is a linker, andwherein n is selected from 1-15 and Tn is an anti-cancer agent, andwherein n is 1(a)-33(a) or 1(b)-4(b) or Tn is 1. The PAC compound can beused in combination with photodynamic therapy and/or radiation therapyto treat a subject with cancer or cancer cells in-vitro.

Another aspect of one embodiment of the present invention provides a PACcompound that when introduced into a cell provides a first cytotoxicagent and a second cytotoxic agent that may work synergistically, forexample to produce a synthetic lethal mutation in the cell.

Another aspect of the present invention provides a method to treat acancer cell in-vitro or in-vivo or tumor in vivo by providing a PACcompound to the cancer cell or tumor, treating the cancer cell or tumorwith one or more of the following: phototherapy of a wavelength of lightto excite the porphyrin of the PAC compound, the sound of the frequencyto activate the porphyrin of the PAC compound, in combination with theanti-cancer agent on the PAC compound.

Another aspect of the present invention provides for a method to treat acancer cell comprising delivering a PAC compound to the cancer cellwherein the porphyrin and the anti-cancer agent act to kill the cancercell via synthetic lethality. In another embodiment, the use of a PACcompound as disclosed herein is provided in the absence ofphenothiazine-derived drug such as chlorpromazine.

In one embodiment of the present invention, the cytotoxic agent (Tn) ofthe general formula Pn-Ln-Tn is not selected from the group consistingof antitumor antibiotics, bleomycin, dactinomycin, mitoxantrone,mitomycin, epipodophyllotoxins, etoposide, teniposide, antimicrotubuleagents, vinblastine, vincristine, vindesine, vinorelbine, other vincaalkaloids, taxanes, paclitaxel (taxol), docetaxel (taxotere), nitrogenmustards, chlorambucil, cyclophosphamide, estramustine, ifosfamide,mechlorethamine, melphalan, aziridines, thiotepa, alkyl sulfonates,busulfan, nitrosoureas, carmustine, lomustine, and streptozocin,platinum complexes, carboplatin cisplatin, alkylators, altretamine,dacarbazine, procarbazine, temozolamide, folate analogs, methotrexate,purine analogs, fludarabine, mercaptopurine, thioguanine, adenosineanalogs, cladribine, pentostatin, pyrimidine analogs, capecitabine,cytarabine, floxuridine, fluorouracil, gemcitabine, substituted ureas,hydroxyurea, camptothecin analogs, irinotecan and topotecan,topoisomerase I inhibitors, and topoisomerase II inhibitors.

In one embodiment of the present invention, the porphyrin (Pn) of thegeneral formula Pn-Ln-Tn is not one of the following: mesoporphyrins,deuteroporphyrins, hematoporphyrins, protoporhyrins, uroporphyrins,coproporphyrins, cytoporphyrins, rhodoporphyrin, pyrroporphyrin,etioporphyrins, phylloporphyrins, heptacarboxyporphyrins,hexacarboxyporphyrins, pentacarboxyporphyrins, and otheralkylcarboxyporphyrins.

In another embodiment the PAC compound does not include one thefollowing combinations: direct conjugation of mesoporphyrin IX todoxorubicin via an amide bond; a conjugate between colchicine-like toxintrilobolide and a tetraaryl zinc porphyrin using a ‘click’ linker;Cytotoxic ruthenium heterocycle conjugated with a porphyrin; the directconjugates of hematoporphyrin IX with platinum drugs such as cisplatinor carboplatin; a direct conjugation of emodin to aryl porphyrins; aconjugation of an aryl porphyrin to retinoic acid via a PEG amidelinker; a direct conjugate of the alkaloid brucine with aryl porphyrins;a conjugate of fluorouracil with meta-phenolic porphyrins using an etherlinkage and direct conjugation to a kinase inhibitor.

Embodiments of the present invention relate to methods of treatingcancer in a subject in need thereof and a PAC compound for the use intreating cancer in a subject in need thereof. Another embodimentprovides for a method of treating cancer cells and a PAC compound forthe use in treating cancer cells in-vitro.

Objects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims (if any).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIG. 1 illustrates TCPP uptake preferentially is cancer cells vs.non-cancer cells.

FIG. 2 illustrates inhibition of TCPP uptake in cancer cells undervarious conditions.

FIG. 3 illustrates a graph of TCPP uptake by a cancer cell treated withdifferent inhibitors of endocytosis.

FIG. 4 illustrates a synthesis scheme for compound OS0026.

FIG. 5 illustrates a synthesis scheme for compound OS0027.

FIG. 6 illustrates a synthesis scheme for compound OS002.

FIG. 7 illustrates a synthesis scheme for compound OS0024.

FIG. 8 illustrates a synthesis scheme for compound OS007.

FIG. 9 illustrates a synthesis scheme for compound OS0030.

FIG. 10 illustrates a synthesis scheme for compound OS0035.

FIG. 11 illustrates a synthesis scheme for compound OS0032.

FIG. 12 illustrates a synthesis scheme for compound OS0025.

FIG. 13 illustrates a synthesis scheme for compound OS0029.

FIG. 14 illustrates a synthesis scheme for compound OS0023.

FIG. 15 illustrates a synthesis scheme for compound OS009.

DETAILED DESCRIPTION OF THE INVENTION

Furthermore, the following terms shall have the definitions set outbelow. It is understood that in the event a specific term is not definedherein below, that term shall have a meaning within its typical usewithin context by those of ordinary skill in the art.

It is to be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

The term “anti-cancer agent” includes cytotoxic agents, which may beselected from antineoplastic and immunomodulating agents such as but notlimited to, 1) Immunosuppressants including: Pomalidomide, Pirfenidone,Lenalidomide, Methotrexate, Thalidomide, Azathioprine; 2) CalcineurinInhibitors including: Voclosporin, Tacrolimus, Ciclosporin,Cyclosporine; 3) Interleukin Inhibitors including: Brodalumab,Siltuximab, Secukinumab, Briakinumab, Canakinumab, Tocilizumab,Mepolizumab, Ustekinumab, Rilonacept, Anakinra, Basiliximab, Daclizumab;4) Tumor Necrosis Factor alpha (tnf-a) Inhibitors including: Golimumab,Certolizumab pegol, Adalimumab, Afelimomab, Infliximab, Etanercept; 5)Selective Immunosuppressants including: Begelomab, Alemtuzumab,Vedolizumab, Apremilast, Teriflunomide, Tofacitinib, Belatacept,Fingolimod, Belimumab, Eculizumab, Abatacept, Natalizumab, Abetimus,Efalizumab, Gusperimus, Everolimus, Alefacept, Leflunomide, Sirolimus,Mycophenolic acid, Antithymocyte immunoglobulin (rabbit), Antilymphocyteimmunoglobulin (horse), Muromonab; 6) Immunostimulants including:Dasiprotimut, Cridanimod, Sipuleucel-T, Plerixafor, Mifamurtide,Histamine dihydrochloride, Glatiramer Acetate, Melanoma vaccine,Tasonermin, Immunocyanin, Thymopentin, Pidotimod, Pegademase, Bcgvaccine, Roquinimex, Lentinan; 7) Interleukins including: Oprelvekin,Aldesleukin; 8) Interferons including: Peginterferon alfa-2a,Peginterferon alfa-2b, Cepeginterferon alfa-2b, Peginterferon beta-1a,Peginterferon beta-1a, Albinterferon alfa-2b, Albumin-interferon alpha,Peginterferon alpha 2a, Peginterferon alpha 2b, Interferon alfacon-1,Interferon beta-1b, Interferon beta-1a, Interferon alfa-n1, InterferonAlfa-2b Recombinant, Interferon Alfa-2a-Recombinant, Interferon gamma,Interferon beta natural, Interferon alfa natural; 9) Colony stimulatingfactors including: Balugrastim, Lipegfilgrastim, Pegfilgrastim,Ancestim, Pegfilgrastim, Ancestim, Lenograstim, Pegfilgrastim,Sargramostim, Molgramostim, Filgrastim; 10) Hormone Antagonists andrelated agents including: Abiraterone, Degarelix, Abarelix, Exemestane,Letrozole, Anastrozole, Formestane, Aminoglutethimide, Enzalutamide,Bicalutamide, Nilutamide, Flutamide, Fulvestrant, Toremifene, Tamoxifen;11) Hormones and Related Agents including: Histrelin, Triptorelin,Goserelin, Leuprolide, Buserelin; 12) Progestogens including:Gestonorone, Medroxyprogesterone, Megestrol; 13) Estrogens including:Fosfestrol, Ethinylestradiol, Ethinyl Estradiol, Polyestradiolphosphate, Diethylstilbestrol; 14) Antineoplastic Agents including:Ixazomib, Belinostat, Sonidegib, Idelalisib, Olaparib, Carfilzomib,Aflibercept, Vismodegib, Panobinostat, Eribulin, Homoharringtonine,Romidepsin, Vorinostat, EG009, Oblimersen, Anagrelide, Celecoxib,Bortezomib, Denileukin diftitox, Arsenic trioxide, Bexarotene,Pegaspargase, Mitotane, Alitretinoin, Irinotecan, Topotecan, Tretinoin,Estramustine, Masoprocol, Miltefosine, Pentostatin, Lonidamine,Hydroxycarbamide, Hydroxyurea, Altretamine, Asparaginase, Amsacrine,Tivozanib, Palbociclib, Cediranib, Nintedanib, Lenvatinib, Ceritinib,Ibrutinib, Cabozantinib, Trametinib, Ponatinib, Dabrafenib, Masitinib,Regorafenib, Ruxolitinib, Axitinib, Crizotinib, Vemurafenib, Bosutinib,Afatinib, Vandetanib, Pazopanib, Everolimus, Temsirolimus, Nilotinib,Lapatinib, Dasatinib, Sorafenib, Sunitinib, Erlotinib, Gefitinib,Imatinib; 15) Sensitizers used in photodynamic/radiation therapyincluding: Efaproxiral, Temoporfin, Aminolevulinic acid, Methylaminolevulinate, Porfimer; 16) Monoclonal antibodies including:Necitumumab, Ramucirumab, Blinatumomab, Pembrolizumab, Nivolumab,Dinutuximab, Obinutuzumab, Ado-trastuzumab emtansine, Pertuzumab,Brentuximab vedotin, Ipilimumab, Ofatumumab, Catumaxomab, Panitumumab,Bevacizumab, Cetuximab, Gemtuzumab, Trastuzumab, Rituximab, Edrecolomab,Methylhydrazines, Procarbazine, Platinum compounds, Polyplatillen,Satraplatin, Oxaliplatin, Carboplatin, Cisplatin; 17) Cytotoxicantibodies and related substances including: Ixabepilone, Mitomycin,Plicamycin, Bleomycin; 18) Anthracyclines and related substancesincluding: Pixantrone, Amrubicin, Valrubicin, Pirarubicin, Mitoxantrone,Idarubicin, Zorubicin, Aclarubicin, Epirubicin, Daunorubicin,Doxorubicin; 19) Actinomycines including: Dactinomycin; 20) PlantAlkaloids and other natural products including: Trabectedin; 21) Taxanesincluding: Cabazitaxel, Docetaxel, Paclitaxel; 22) ColchicineDerivatives including: Demecolcine; 23) Podophyllotoxin derivativesincluding Teniposide, Etoposide; 24) Vinca alkaloids and analoguesincluding: Vintafolide, Vinflunine, Vinorelbine, Vindesine, Vincristine,Vinblastine; 25) Pyrimidine analogues including: Trifluridine, Tegafur,Fluorouracil, Decitabine, Azacitidine, Capecitabine, Gemcitabine,Carmofur, Tegafur, Fluorouracil, Cytarabine; 26) Purine analoguesincluding: Nelarabine, Clofarabine, Fludarabine, Cladribine, Tioguanine,Mercaptopurine, Pralatrexate, Pemetrexed, Raltitrexed, Methotrexate; 27)Other alkylating agents including: Dacarbazine, Temozolomide,Pipobroman, Mitobronitol; 28) Epoxides including: Etoglucid; 29)Nitrosoureas including: Ranimustine, Nimustine, Fotemustine,Streptozocin, Semustine, Lomustine, Carmustine; 30) Ethylene iminesincluding: Carboquone, Triaziquone, Thiotepa; 31) Alkyl sulfonatesincluding: Mannosulfan, Treosulfan, Busulfan; 32) Nitrogen mustardanalogues including: Bendamustine, Prednimustine, Trofosfamide,Ifosfamide, Mechlorethamine, Melphalan, Chlorambucil, Cyclophosphamideand derivatives and analogs of the cytotoxic agents disclosed. Theantineoplastic and immunomodulating agents may be classified asmicrotubule-stabilizing agents, microtubule-disruptor agents, alkylatingagents, antimetabolites, epidophyllotoxins, antineoplastic enzymes,topoisomerase inhibitors, inhibitors of cell cycle progression,antisense molecules, toxins and platinum coordination complexes.

As used herein, an “anticancer therapeutic effect” includes one or moreof the following: inhibition of cancer cell growth, increased cancercell death (a tumoricidal reaction), reduction in tumor invasiveness,reduction in overall tumor burden, reduction in local tumor burden,reduction in size of the primary tumor, prevention of metastases,reduction in the number of metastases, reduction in the size ofmetastases, and prolonged life. While it is desired that the treatmentrender the subject free of disease, it is not intended that the presentinvention be limited to curing cancer. There is therapeutic benefit evenif the cancer is simply slowed in its progression. It is not intendedthat the present invention be limited to the magnitude of the effect.For example, the reduction in size of the primary tumor (or ofmetastases) can be as little as a 10% reduction or as great as a 90%reduction (or more). It is also not intended that the present inventionbe limited to the duration of the anticancer therapeutic effect. Thetreatment (using the various embodiments described herein) may result inonly temporary inhibition of cancer cell growth, temporary increasedcancer cell death, temporary reduction in tumor invasiveness, temporaryreduction in overall tumor burden, temporary reduction in local tumorburden, temporary reduction in size of the primary tumor, temporaryprevention of metastases, temporary reduction in the number ofmetastases, or temporary reduction in the size of metastases. Thetemporary effects may last weeks to months, or months to years. Theseparameters are relatively easy to measure (e.g., by monitoring the sizeof the primary tumor(s) and metastases over time). With respect toprevention of metastases and prolonging life, these parameters may bemeasured against patient population data for the particular tumor type,stage, and the like. As used herein, a “cancer preventative effect” or“protective effect” comprises an effect that reduces the incidence ofnew cancers. This parameter can be proved in animals and measured inhumans on a population basis.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a cytotoxic agent administered to a patient.Bioavailability is an absolute term that indicates measurement of boththe time (rate) and total amount (extent) of drug that reaches thegeneral circulation from an administered dosage form.

The term “cytotoxic activity” refers to a cell-killing, a cytostatic oran anti-proliferative effect of a porphyrin anti-cancer agent conjugateor an intracellular metabolite of a Drug Linker Ligand conjugate.Cytotoxic activity may be expressed as the IC₅₀ value in cells, which isthe concentration (molar or mass) per unit volume at which half thecells die and/or the IC₁₀ value in animals, which is the concentration(molar or mass) per unit volume at which 10% of the animals die. Lightinduced porphyrin cytotoxicity occurs upon activation of porphyrin.

Cancers are classified in two ways: by the type of tissue in which thecancer originates (histological type) and by primary site, or thelocation in the body where the cancer first developed. From ahistological standpoint there are hundreds of different cancers, whichare grouped into six major categories: Carcinoma, Sarcoma, Myeloma,Leukemia, Lymphoma, Mixed Types, Central Nervous System and Mesotheliomaas identified from the world wide web cancer research society websitecrs-src.ca last visited on May 5, 2016.

The term “cancer” is used throughout the specification to refer to acell(s) possessing one or more of the following abnormal growthcharacteristics: uncontrolled proliferation, immortality, metastaticpotential, rapid growth and proliferation rate, perturbed oncogenicsignaling, and certain morphological characteristic features and mayoriginate from: epithelial cell tissue (carcinomas), blood cells, bonemarrow, and immune cells (leukemias, lymphomas, myelomas), connectivetissue, bone, cartilage, fat, muscle, blood vessels (sarcomas), centralnervous system tissue, glial or supportive cells (gliomas, blastomas CNSlymphoma), mesothelium lining (mesothelioma of lung, heart, abdominalcavity), melanoma (mesodermal origin). As used herein, the term canceris used to describe all cancerous disease states applicable to diagnosisand treatment according to the present invention and embraces orencompasses the pathological process associated with virtually allcancers types, including carcinomas, sarcoma, myeloma, leukemia,lymphoma, mixed types. In a preferred embodiment, the cancer is a solidtumor.

Examples of cancers which may be diagnosed/treated using compounds andmethods according to the present invention include, but is not limitedto, carcinomas (e.g., squamous-cell carcinomas, adenocarcinomas,hepatocellular carcinomas, and renal cell carcinomas), particularlythose of the bladder, bowel, breast, cervix, colon, esophagus, head,kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; andmalignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin'slymphoma; and malignant melanomas; myeloproliferative diseases;sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi'ssarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, andsynovial sarcoma; tumors of the central nervous system (e.g., gliomas,astrocytomas, oligodendrogliomas, ependymomas, glioblastomas,neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas,pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, andSchwannomas); germ-line tumors (e.g., bowel cancer, breast cancer,prostate cancer, cervical cancer, uterine cancer, lung cancer, ovariancancer, testicular cancer, thyroid cancer, astrocytoma, esophagealcancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer,and melanoma); mixed types of neoplasias, particularly carcinosarcomaand Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumorand teratocarcinomas. For example Adrenal Cancer, Anal Cancer, Bile DuctCancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors In Adults,Brain/CNS Tumors In Children, Breast Cancer, Breast Cancer In Men,Cancer in Adolescents, Cancer in Children, Cancer in Young Adults,Cancer of Unknown Primary, Castleman Disease, Cervical Cancer,Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Ewing FamilyOf Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal CarcinoidTumors, Gastrointestinal Stromal Tumor (GIST), Gestational TrophoblasticDisease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal andHypopharyngeal Cancer, Liver Cancer, Lung Cancer, Lung Cancer—Non-SmallCell, Lung Cancer—Small Cell, Lung Carcinoid Tumor, Lymphoma, Lymphomaof the Skin, Malignant Mesothelioma, Multiple Myeloma, MyelodysplasticSyndrome, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma InChildren, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, OvarianCancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, ProstateCancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoma—Adult Soft Tissue Cancer, Skin Cancer, Skin Cancer—Basal andSquamous Cell, Skin Cancer—Melanoma, Skin Cancer—Merkel Cell, SmallIntestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer,Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer,Waldenstrom Macroglobulinemia, Wilms Tumor may be the subject oftreatment with one or more of the compositions as disclosed herein.

The term “compound”, as used herein, unless otherwise indicated, refersto any specific chemical compound disclosed herein. In certaininstances, the term may also refer to stereoisomers and/or opticalisomers (including racemic mixtures) or enantiomerically enrichedmixtures of disclosed compounds. In certain instances, the term may alsorefer to salts, metabolites, prodrugs, crystals, polymorphs, analogues,solvates and hydrates.

It should be recognized that compounds referred to herein can containchiral carbon atoms. In other words, it may have optical isomers ordiastereoisomers. Compounds may also include salts and their polymorphs.Further a composition may exist as any combination of the compounds.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits the function of cells and/or causes destruction of cellswherein the term cytotoxic agents includes cytostatic agents. The termis intended to include radioactive isotopes (e.g., ²¹¹At, ¹³¹I, ¹²⁵I,⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, and radioactive isotopes ofLu), and toxins such as small molecule toxins or enzymatically activetoxins of bacterial, fungal, plant or animal origin, including syntheticanalogs and derivatives thereof. In one aspect, the term does notinclude a radionuclide.

A cytotoxic agent may be covalently appended to porphyrins with asuitable linker and include solubilizing linkers which include forexample polyethylene glycol (PEG) moieties but not limited thereto andwherein the cytotoxic agent in one embodiment include those identifiedin Table 6, Table 7 and Table 8 and dolastatins, e.g., dolastatin 10,dolastatin 15, monomethylauristatin E, monomethylauristatin F,tasidotin, cemadotin.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or composition which, in context, isused to produce an intended result, whether that result relates to thetreatment of a cancer in a patient or subject or with cells in-vitro.The term effective subsumes all other effective amount or effectiveconcentration terms, which are otherwise described or used in thepresent application. In the case of cancer, the effective amount of thecomposition may reduce the number of cancer cells; reduce the tumorsize; reduce the number of tumor sites, inhibit (i.e., slow to someextent or stop) cancer cell infiltration into adjacent and or distaltissues.

The term “linker” is used throughout the specification within context todescribe a covalent moiety that attaches a porphyrin and an anti-canceragent, for example covalently. A wide variety of linkers can be used,and according to one embodiment, the invention is not limited by thetype of linker used. Examples of linkers include, but are not limitedto, substituted and unsubstituted C₁-C₁₂ alkyl, alkenyl, and alkynylgroups, C₁-C₁₂ heteroalkyl, heteroalkenyl, and heteroalkynyl groups, andC₆-C₂₀ aryl-containing and heteroaryl-containing linking groups andL9-L16.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, especially including adomesticated mammal and preferably a human, to whom a treatment orprocedure is performed. For treatment of those conditions or diseasestates which are specific for a specific animal such as a human patient,the term patient refers to that specific animal. In most instances, thepatient or subject of the present invention is adomesticated/agricultural animal or human patient of either or bothgenders.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the art.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsfrom which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases. Inorganic bases from which salts can bederived include, for example, sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike. Organic bases from which salts can be derived include, forexample, primary, secondary, and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines, basicion exchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,trifluoroacetyl and ethanolamine. In some embodiments, thepharmaceutically acceptable base addition salt is chosen from ammonium,potassium, sodium, calcium, and magnesium salts.

The term “alkyl” refers to saturated aliphatic groups includingstraight-chain, branched-chain, cyclic groups, and combinations thereof,having the number of carbon atoms specified, or if no number isspecified, having up to 12 carbon atoms. “Straight-chain alkyl” or“linear alkyl” groups refer to alkyl groups that are neither cyclic norbranched, commonly designated as “n-alkyl” groups. Examples of alkylgroups include, but are not limited to, groups such as methyl, ethyl,n-propyl, isopropyl, butyl, n-butyl, isobutyl, sec-butyl, t-butyl,pentyl, n-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andadamantyl. Branched-chain groups would include, but not be limited to,isopropyl, isobutyl, neopentyl, tertiary butyl and tertiary amyl. Cyclicgroups can consist of one ring, including, but not limited to, groupssuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,or multiple fused rings, including, but not limited to, groups such asadamantyl or norbornyl. Preferred subsets of alkyl groups includeC₁-C₁₂, C₁-C₁₀, C₁-C₈, C₁-C₆, C₁-C₄, C₁-C₂, C₃-C₄, C₃, and C₄ alkylgroups.

The term “Aryl” or “Ar” refers to an aromatic carbocyclic group having asingle ring (including, but not limited to, groups such as phenyl, andincludes both unsubstituted and substituted aryl groups. “Substitutedaryls” refers to aryls substituted with one or more substituents,including, but not limited to, groups such as alkyl, halogen, alkoxy(OR), amino or alkylamino (NH₂, NHR, NRR′), hydroxyl, carboxy, phenyl,cyano, carboalkoxy and carboxamide, or a functionality that can besuitably blocked, if necessary for purposes of the invention, with aprotecting group.

The term “alkoxy” as used herein refers to an alkyl group linked to anoxygen atom and having the number of carbon atoms specified, or if nonumber is specified, having up to 12 carbon atoms. Examples of alkoxygroups include, but are not limited to, groups such as methoxy, ethoxy,and t-butoxy.

The terms “halo” and “halogen” as used herein refer to Cl, Br, F or Isubstituents.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on a PAC compound,whereby the linker, the part of the compound that connects the cytotoxicagent and the porphyrin is broken, resulting in the free cytotoxicagent, or other metabolite of the conjugate dissociated from theporphyrin inside the cell. The cleaved moieties of PAC compounds arethus intracellular metabolites.

In addition to the treatment of cancers as described above, the presentinvention also may be used preferably to treat cancers such aschoriocarcinoma, testicular choriocarcinoma, non-seminomatous germ celltesticular cancer, placental cancer (trophoblastic tumor) and embryonalcancer, among others. In a preferred embodiment, the cancer to betreated is lung cancer.

Formulations containing the compounds according to the present inventionmay take the form of liquid, solid, semi-solid or lyophilized powderforms, such as, for example, solutions, suspensions, emulsions,sustained-release formulations, tablets, capsules, powders,suppositories, creams, ointments, lotions, aerosols, patches or thelike, preferably in unit dosage forms suitable for simple administrationof precise dosages.

Pharmaceutical compositions according to the present invention typicallyinclude a conventional pharmaceutical carrier or excipient and mayadditionally include other medicinal agents, carriers, adjuvants,additives and the like. Excipients include those identified on the worldwide web at fda.gov and equivalents thereto as of the date of the filingof this application. The weight percentage ratio of theanti-cancer/porphyrin to the one or more excipients can be between about20:1 to about 1:60, or between about 15:1 to about 1:45, or betweenabout 10:1 to about 1:40, or between about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2:1 or 1:1 to about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,1:15, 1:20, 1:25, 1:30, or 1:35, and preferably is about 20:1, 19:1,18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1or 5:1. In some embodiments, formulations of the invention comprisebetween about 250 mg to about 500 mg, or between about 300 mg to about450 mg, or about 325 mg to about 425 mg of total porphyrin/anti-canceragent and may optionally contain one or more suitable pharmaceuticalexcipients.

An injectable composition for parenteral administration (e.g.intravenous, intramuscular intrathecal, intraperitoneal or intracranial)will typically contain the compound in a suitable i.v. solution, such assterile physiological salt solution. The compound may also be formulatedas a suspension in an aqueous emulsion. A composition comprises a PACcompound or a pharmaceutically acceptable salt thereof and at least onpharmaceutically acceptable carrier.

Liquid compositions can be prepared by dissolving or dispersing thepharmaceutical composition comprising the PAC compound and optionalpharmaceutical adjuvants, in a carrier, such as, for example, aqueoussaline, aqueous dextrose, glycerol, or ethanol, to form a solution orsuspension. For use in an oral liquid preparation, the composition maybe prepared as a solution, suspension, emulsion, or syrup, beingsupplied either in liquid form or a dried form suitable for hydration inwater or normal saline.

For oral administration, such excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. If desired, the composition may also contain minor amounts ofnon-toxic auxiliary substances such as wetting agents, emulsifyingagents, or buffers.

When the composition is employed in the form of solid preparations fororal administration, the preparations may be tablets, granules, powders,capsules or the like. In a tablet formulation, the composition istypically formulated with additives, e.g. an excipient such as asaccharide or cellulose preparation, a binder such as starch paste ormethyl cellulose, a filler, a disintegrator, and other additivestypically used in the manufacture of medical preparations.

Methods for preparing such dosage forms are known or are apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (17th Ed., Mack Pub. Co. 1985). The composition to beadministered will contain a quantity of the selected compound in apharmaceutically effective amount for therapeutic use in a biologicalsystem, including a patient or subject according to the presentinvention.

Methods of treating patients or subjects in need, for a particulardisease state comprise administration of an effective amount of apharmaceutical composition comprising therapeutic amounts ofporphyrin/anti-cancer agent described herein and optionally at least oneadditional bioactive (e.g. anti-cancer) agent according to the presentinvention. The amount of porphyrin/anti-cancer agent used in the methodsof treatment of the instant invention that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated, the particular mode of administration. Forexample, the compositions could be formulated so that a therapeuticallyeffective dosage of between about 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 mg/kg ofpatient/day or in some embodiments, greater than 100, 110, 120, 130,140, 150, 160, 170, 180, 190 or 200 mg/kg of the novelporphyrin/anti-cancer agent can be administered to a patient receivingthese compositions.

In one embodiment, a compound of Formula III

or a pharmaceutically acceptable salt, enantiomer, solvate, or polymorphthereof is provided, wherein A₁-A4 each are independently selected froma substituted aromatic ring or substituted heteroaromatic ring. B₁represents a covalent linker moiety which connects A₁ to a cytotoxicagent (also referred to herein as “cytotoxin”) Z₁.

In a specific embodiment, A₁ represents an aromatic ring bearing acarboxylic amide functional group, where the position of the carboxylicamide may be ortho, meta or para with respect to the porphyrin ring andwherein the C—N bond of the carboxamide functional group serves tocovalently connect A₁ to linker B₁. Further, A2, A3 and A4 representmono-substituted aromatic rings wherein the substituent mayindependently occupy the ortho, meta or para position with respect tothe porphyrin ring. The substituents on A2, A3 and A4 may independentlybe lower alkyl, branched lower alkyl, cycloalkyl, halogens (F, Cl, Br,I), cyano, amino or substituted amino, sulfonyl (including sulfonicacids, esters or amides), phenol, aromatic ethers (OR, where R is loweralkyl) or carbonyl (including carboxylic acids, carboxylic esters (COOR,where R is lower alkyl) or carboxylic amides. B₁ can be selected fromthe group L11-L14 (wherein B₁ is defined as the chemical formula betweenA₁ and Z₁ of Table 1). The group Z₁ may be a toxin of the anthracyclinetype (Table 2), kinase inhibitor (Table 3) or auristatin type (Table 4),respectively, bound to B₁ and wherein the a nitrogen atom of thecytotoxin Z₁ is part of a carboxamide functional group connecting B₁ toZ₁.

In a preferred embodiment, A₁ represents an aromatic ring bearing acarboxylic amide (carboxamide) functional group, where the position ofthe carboxylic amide may be ortho, meta or para with respect to theporphyrin ring. Further, A2, A3 and A4 represent mono-substitutedaromatic rings wherein the substituent is a carboxylic acid, carboxylicester or carboxylic amide and may occupy the ortho, meta or paraposition with respect to the porphyrin ring. B₁ may be selected from thegroup L11 or L13 (Table 1) Z₁ may be selected from the cytotoxins of theanthracycline, kinase inhibitor or auristatin type, respectively (Table2-4).

In a most preferred embodiment, A₁ represents an aromatic ring bearing acarboxylic amide functional group, where the position of the carboxylicamide is para with respect to the porphyrin ring. Further, A2, A3 and A4represent mono-substituted aromatic rings wherein the substituent is acarboxylic acid, or carboxylic methyl ester in the para position withrespect to the porphyrin ring. B₁ may be selected from the group L11 orL13 (Table 1). Z₁ may be selected from the toxins T1b, 1 or T4c, inTable 2-4, respectively.

TABLE 1 B₁ formulas L11

L12

L13

L14

(n = 1-12)

TABLE 2 Cytotoxins Z₁ (anthracycline type formulas)

Tnb R1 R2 * T1b OH OMe R T2b H OMe R T3b H H R T4b OH OMe S

TABLE 3 Cytotoxins Z₁ (kinase inhibitor type formulas) Parent toxinToxin Structure name 1

Crizotinib T1a

entrecitinib T3a

pelitinib T4a

lapatinib T8a

bosutinib T10a

imatinib T14a

vandetanib T15a

bosutinib 18a

sunutinib T19a

ponatinib T21a

masitinib T27a

nintedanib T29a

ceritinib T31a

palbociclib T32a

osimertinib T33a

Olmutinib

TABLE 4 CytoToxins Z₁ (auristatin type formula)

 T4c

 T5c

 T9c

T10c

In another specific embodiment, A₁ represents an aromatic ring bearingan aromatic ether functional group, where the position of the aromaticether may be ortho, meta or para with respect to the porphyrin ring.Further, A2, A3 and A4 represent mono-substituted aromatic rings whereinthe substituent may independently occupy the ortho, meta or paraposition with respect to the porphyrin ring. The substituents on A2, A3and A4 may independently be lower alkyl, branched lower alkyl,cycloalkyl, halogens (F, Cl, Br, I), cyano, amino or substituted amino,sulfonyl (including sulfonic acids, esters or amides), phenol oraromatic ethers (OR, where R is lower alkyl) or carbonyl (includingcarboxylic acids, carboxylic esters (COOR, where R is lower alkyl) orcarboxylic amides). B₁ may be selected from the group L9, L10, L15, L16(Table 5). Z₁ may be selected from the cytotoxins of the anthracycline,kinase inhibitor or auristatin type, respectively (Tables 2-4).

In a preferred embodiment, A₁ represents an aromatic ring bearing anaromatic ether functional group, where the position of the aromaticether may be ortho, meta or para with respect to the porphyrin ring.Further, A2, A3 and A4 represent mono-substituted aromatic rings whereinthe substituent is a phenol and may occupy the ortho, meta or paraposition with respect to the porphyrin ring. B₁ may be selected from thegroup L9 or L15 (Table 5). Z₁ may be selected from the cytotoxins of theanthracycline, kinase inhibitor or auristatin type, respectively (Tables2-4).

In a most preferred embodiment, A₁ represents an aromatic ring bearingan aromatic ether functional group, where the position of the aromaticether is meta with respect to the porphyrin ring. Further, A2, A3 and A4represent mono-substituted aromatic rings wherein the substituent is aphenol in the meta position with respect to the porphyrin ring. B₁ is L9(Table 5). Z₁ may be selected from the cytotoxins T1b or 1 Tables 2-3,respectively.

TABLE 5 B₁ Structures L9 

L10

L15

L16

(chemical formula represented between A₁ and Z₁ where n = 1-12)

In another specific embodiment, A₁ represents a heteroaromatic ringbearing an alkylated nitrogen atom (i.e., a pyridinium) where theposition of the pyridinium nitrogen may be in the 2, 3 or 4 positionwith respect to the porphyrin ring. Further, A2, A3 and A4 representpyridine, or alkylpyridinium, rings where the position of the pyridine,or alkylpyridinium, nitrogen may independently be in the 2, 3 or 4position with respect to the porphyrin ring. The alkyl portion of thealkylpyridinium moiety represents lower alkyl or branched alkyl. B₁ maybe selected from the group L9, L10, L15, L16 (Table 5). Z₁ may beselected from the cytotoxins of the anthracycline, kinase inhibitor orauristatin type, respectively (Tables 2-4).

In a most preferred embodiment, A₁ is a heteroaromatic ring bearing anitrogen atom (i.e., a pyridinium) where the position of the pyridiniumnitrogen is in the 4 position with respect to the porphyrin ring.Moreover, A2, A3 and A4 represent pyridine rings wherein the position ofeach nitrogen atom is in the 4 position relative to the porphyrin ring.B₁ may be selected from the group L9 or L15 (Table 5). Z₁ may beselected from the cytotoxins T1b or 1, in Tables 2 and 3, respectively.

EXAMPLES

Porphyrins preferentially taken up by cancer cells as compared tonon-cancer cells. The mechanism responsible for this is poorlyunderstood. Table 9 is a list of some of the porphyrins and the type ofcancer cells that exhibit preferential uptake of the porphyrin ascompared to uptake of the porphyrin by non-cancer cells. The list is notexhaustive but illustrative of the effect.

TABLE 9 CANCER CELL LINES Porphyrin Model hematoporphyrin Murinecarcinoma L-aspartyl-chlorin e6 Murine carcinoma Chloraluminum Murinecarcinoma pthalocyanine 5,10,15,20-tetrakis Human bladder cancer (T24)cells in (5-morpholinopentyl)- murine xenograft 21H,23H-Porphin (MPP)Venteporphyrin Human colorectal cancer cells Protoporphyrin IX Humansquamous cell carcinoma cells Porfimer sodium Tumor-normal tissueselectivity in human patients Lemuteporfin (a chlorin) Selectiveaccumulation in mitogen- activated lymphoid cells Metalloporphyrins andHuman colon and sarcoma cells conjugates tumor accumulation.Sulfonylated aryl Human melanoma cells vs. porphyrins fibroblasts(normal cells) Mn pyridylporphyrins Selective toxicity against humanbreast cancer cells vs. normal breast cells. Temoporfin squamous cellcarcinoma of the head and neck

Porphyrin tetrakis(4-carboxyphenyl)porphyrin (TCPP) uptake by a panel ofhuman lung cancer cell lines including, HCC15, H157, and H358 wereexamined using flow cytometry in under varying conditions to manipulateclathrin-dependent and independent endocytosis by chemical inhibitors.Referring now to FIG. 1 , TCPP uptake in cancer cells (HCC15) ascompared to bone marrow cells in a dose dependent manner is illustrated.Similar results are observed with mouse normal lung cells as is shownfor bone marrow cells. The uptake of TCPP by HCC15 is greater than2-fold at 10 ug/ml as compared to the non-cancer cells. As isillustrated in FIG. 2 , TCPP uptake in cancer cell lines HCC15, H157 andH358 is moderately inhibited by sucrose and nearly completely inhibitedby cold temperature, suggesting that endocytosis is at play in TCPPuptake by the cell. Chlorpromazine, an antagonist of clathrin-mediatedendocytosis, inhibited TCPP uptake in a cancer cell line by up to 80% asis illustrated in FIG. 3 . In contrast, the clathrin-independentendocytosis inhibitor filipin had no effect on TCPP uptake. It has beenspeculated that preferential porphyrin uptake by cancer cells ascompared to non-cancer cells is facilitated by the increased number ofLDL receptor (LDLR) on the surface of cancer cells. To examine LDLRcontribution on TCPP uptake, lung cancer cells were manipulated toexpress no LDLR, which reduced TCPP uptake by only 20%. Surprisingly,TCPP uptake in human fibroblasts without functional LDLR showed noinhibition of TCPP at all. These data suggest that additional receptors(to LDLR) and/or mechanisms (to endocytosis) are involved in TCPPuptake.

PAC compounds of Formula III can be synthesized as described below. Theprocedure for the synthesis of target OS0026 is illustrated in FIG. 4 .

STEP 1: Synthesis of1-(5-methoxy-5-oxopentyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-iumbromide (3). A mixture of meso-tetrakis(4-pyridyl)porphyrin (1) (420 mg,0.67 mmol) and methyl 5-bromopentanoate 2 (1.58 g, 8.08 mmol) in 33 mLEtOH and 100 mL chloroform was stirred at reflux for 6 days. Thereaction mixture was purified by two successive column chromatographyover silica gel using EtOH/Chloroform (3/7) as eluent to give 3 as apurple solid (186 mg, 30%). ¹H-NMR (300 MHz, DMSO-d₆): δ 11.95 (s, 2H),9.52-9.55 (d, 2H), 8.94-9.10 (m, 16H), 8.29-8.31 (t, 6H), 4.94 (t, 2H),3.68 (s, 3H), 2.51-2.58 (m, 2H), 2.66 (m, 2H), 1.84 (m, 2H). MS m/z=733[M]⁺. Purity by HPLC: >95%, t_(R)=3.48.

STEP 2: Synthesis of1-(4-carboxybutyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-iumchloride (4). Ester derivative 3 (230 mg, 028 mmol) was dissolved in 46mL 1N HCl to give a green solution, which was stirred at reflux for 3 h.The reaction mixture was lyophilized to afford the corresponding acidderivative 4 as a purple solid (244 mg, crude yield 100%). The crudeproduct was used directly for the next step without furtherpurification. ¹H-NMR (300 MHz, DMSO-d₆): δ 11.90 (s, 2H), 9.50 (d, 2H),9.35-9.37 (d, 6H), 9.13 (m, 8H), 9.02-9.04 (d, 2H), 8.79 (s, 6H), 5.5(br, 5H), 5.0 (m, 2H), 2.3 (m, 2H), 1.85 (m, 2H). Purity by HPLC: >95%,t_(R)=3.29.

STEP 3: Synthesis of1-(5-(((2S,3S,4R,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)amino)-5-oxopentyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-iumTFA salt (OS0026). To a solution of acid derivative 4 (45 mg, 0.05 mmol)in 4.5 mL DMF was added HATU (45 mg, 0.12 mmol) in one portion, followedby the dropwise addition of DIPEA (45 mg, 0.35 mmol) at roomtemperature. The reaction mixture was stirred for 5 min at r.t beforethe addition of doxorubicin hydrochloride (30 mg, 0.056 mmol) in oneportion. The reaction mixture was stirred at room temperature overnightand evaporated to remove solvent. The residue was purified by reversephase preparatory HPLC using ACN/water with TFA as eluent to afford thedesired target compound OS0026 (30 mg, 33%) as a purple solid. Purity byHPLC: >95%, t_(R)=3.48.

HPLC Condition.

Agilent Tech 1100 series HPLC System equipped with Variable WavelengthDetector and ELSD Detector

Column: Agela, Durashell C18, 3.0 μm, 4.60×50 mm.

Mobile Phase: A ACN with 0.1% TFAMobile Phase: D H₂O with 0.1% TFA

Gradient

Time A (ACN with 0.1% TFA) D (H₂O with 0.1% TFA) 0 5 95 5.75 95 5 8 95 59 5 95

Detection: UV at 254 nm & ELSD

Flow rate: 1 mL/minInjection volume: 5 μL

Column Temp: RT

Run time: 9 min

The synthetic scheme for OS0026 illustrates the alkylation ofmeso-tetrakis(4-pyridyl)porphyrin (1) with methyl 5-bromopentanoate (2)to yield an intermediate ester (3), which is then hydrolyzed to thecorresponding acid (4) and subsequently coupled to the aminosugarnitrogen of doxorubicin using the peptide coupling reagent HATU toafford OS0026.

Further, a pyridyl porphyrin conjugate having the general structure P2

wherein X is nitrogen (N) is illustrated. However, N may alternativelybe positioned on each ring at Y or Z instead of X with the linker boundto the N at the alternative position. Thus, on each pyridine ring of P2,N may be independently positioned at either X or Y or Z, with CH groupsoccupying the remaining two positions. For example, the porphyrinmeso-tetrakis(3-pyridyl) porphyrin could be represented as Y is N, X andZ are CH on all four pyridine rings. In a general embodiment, theposition of the N atom on each ring may differ. In a more preferredembodiment, the position of the N atom in each pyridine ring is the samein all four pyridine rings. In a preferred embodiment, the porphyrinmeso-tetrakis(4-pyridyl) porphyrin could be represented as X=N, and Yand Z are CH on all four pyridine rings.

P2 or an isomer thereof is reacted with an alkylator selected fromeither L1 or L2. In particular, n is 1-12 for L1 or L2 and X is aleaving group suitable for an S_(N)2 reaction with a pyridine nitrogento form an alkylpyridinium salt (e.g., 3 in FIG. 4 ). In this context,the leaving group X is either a halogen leaving groups (Cl, Br, I) or avariety of activated sulfonyl esters such as mesylates, tosylates ortriflates. Moreover, Y on L1 or L2 may be a variety of carboxylateesters (OR), wherein R may be chosen to be H, lower straight chain orbranched alkyl, cycloalkyl, aryl or heteroaryl. In a preferredembodiment, L1 is selected such that n=3, X is bromo and Y is methoxy.

The toxin conjugated in FIG. 4 is doxorubicin, but could more generallybe chosen from the anthracycline antibiotics possessing an aminosugarmoiety capable of forming an amide bond, as illustrated in Table 6,wherein doxorubicin is illustrated as T1b and examples of analoguestherefore are illustrated at T2b-T4b. In a preferred embodiment, P2 isselected such that X=N and Y and Z are both CH. Moreover, X ispositioned at the 4-position relative to the porphyrin ring. In thisembodiment, L1 is selected such that X is Br and Y is OMe, n=3, and thetoxin is T1b.

TABLE 6 Examples of anthracyclines containing an aminosugar

Tnb R1 R2 * T1b OH OMe R T2b H OMe R T3b H H R T4b OH OMe S

The procedure for the synthesis of target OS0027 is illustrated in FIG.5 and described below.

STEP 1: Synthesis of1-(5-methoxy-5-oxopentyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-iumbromide (3). A mixture of meso-tetrakis(4-pyridyl)porphyrin (1) (1) (420mg, 0.67 mmol) and methyl 5-bromopentanoate 2 (1.58 g, 8.08 mmol) in 33mL EtOH and 100 mL chloroform was stirred at reflux for 6 days. Thereaction mixture was purified by two successive column chromatographyover silica gel using EtOH/Chloroform (3/7) as eluent to give 3 as apurple solid (186 mg, 30%). ¹H-NMR (300 MHz, DMSO-d₆): δ 11.95 (s, 2H),9.52-9.55 (d, 2H), 8.94-9.10 (m, 16H), 8.29-8.31 (t, 6H), 4.94 (t, 2H),3.68 (s, 3H), 2.51-2.58 (m, 2H), 2.66 (m, 2H), 1.84 (m, 2H). MS m/z=733[M]⁺. Purity by HPLC: >95%, t_(R)=3.48.

STEP 2: Synthesis of1-(4-carboxybutyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-iumchloride salt (4). Ester derivative 3 (230 mg, 0.28 mmol) was dissolvedin 46 mL 1N HCl to give a green solution, which was stirred at refluxfor 3 h. The reaction mixture was lyophilized to afford thecorresponding acid derivative 4 as a purple solid (244 mg, crude yield100%). The crude product was used directly for the next step withoutfurther purification. ¹H-NMR (300 MHz, DMSO-d₆): δ 11.90 (s, 2H), 9.50(d, 2H), 9.35-9.37 (d, 6H), 9.13 (m, 8H), 9.02-9.04 (d, 2H), 8.79 (s,6H), 5.5 (br, 5H), 5.0 (m, 2H), 2.3 (m, 2H), 1.85 (m, 2H). Purity byHPLC: >95%, t_(R)=3.29.

Synthesis of(5)-1-(5-(4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-5-oxopentyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-iumTFA salt (OS0027). To a solution of acid derivative 4 (86 mg, 0.1 mmol)in 8.6 mL DMF was added HATU (52 mg, 0.23 mmol) in one portion, followedby the dropwise addition of DIPEA (86 mg, 0.67 mmol) at roomtemperature. The resulted mixture was stirred for 5 min at r.t beforethe addition of crizotinib hydrochloride (52 mg, 0.11 mmol) in oneportion. The resulted mixture was stirred at room temperature overnightand evaporated to remove solvent. The residue was purified by reversephase preparatory HPLC using ACN/Water with TFA as eluent to afford thedesired target compound OS0027 as a purple solid (27 mg, 17%). MS (ESI)m/z=1150 [M]⁺. Purity by HPLC (USD): >95%, t_(R)=4.00.

HPLC Condition.

Agilent Tech 1100 series HPLC System equipped with Variable WavelengthDetector and ELSD Detector

Column: Agela, Durashell C18, 3.0 μm, 4.60×50 mm.

Mobile Phase: A ACN with 0.1% TFAMobile Phase: D H₂O with 0.1% TFA

Gradient

Time A (ACN with 0.1% TFA) D (H₂O with 0.1% TFA) 0 5 95 5.75 95 5 8 95 59 5 95

Detection: UV at 254 nm & ELSD

Flow rate: 1 mL/minInjection volume: 5 μL

Column Temp: RT

Run time: 9 min

The synthetic scheme for OS0027 illustrates the alkylation ofmeso-tetrakis(4-pyridyl)porphyrin (1) methyl 5-bromopentanoate (2) toyield an intermediate ester (3), which is then hydrolyzed to thecorresponding acid (4) and subsequently coupled to the secondary amineof the toxin (crizotinib) using the peptide coupling reagent HATU toafford OS0027.

In one embodiment, a pyridyl porphyrin having the general structure ofP2 is used. P2, having a nitrogen atom (N) is positioned at X or Y or Z,with CH occupying the remaining two positions. For example, theporphyrin meso-tetrakis(3-pyridyl)porphyrin could be represented as Y isN, X and Z are CH on all four pyridine rings. In a general embodiment,the position of the N on each ring may differ. In a preferredembodiment, the position of the N on each ring is the same.

In another general embodiment, P2 is reacted with an alkylator selectedfrom either L1 or L2. In L1 or L2, n is 1-12, X is a leaving groupsuitable for an S_(N)2 reaction with a pyridine nitrogen atom to form analkylpyridinium species (e.g., 3 in FIG. 5 ). In this context, theleaving group X is either a halogen leaving groups (Cl, Br, I) or avariety of activated sulfonyl esters such as mesylates, tosylates ortriflates. Moreover, Y on L1 or L2 may be a variety of carboxylateesters (OR), wherein R may be chosen to be H, lower straight chain orbranched alkyl, cycloalkyl, aryl or heteroaryl. In a preferredembodiment, X is bromo and Y is methoxy.

The specific toxin illustrated in FIG. 5 is kinase inhibitor crizotinib(1, Table 7). In an embodiment, crizotinib is replaced with the kinaseinhibitors Tna (n=1-33) in Table 7. In this context, X represents thelocation of amide bond formation to L1 or L2. In a preferred embodiment,P2 is selected such that X=N and Y and Z are both CH. Moreover, X ispositioned at the 4-position relative to the porphyrin ring. In thisembodiment, L1 is selected such that X is Br and Y is OMe, n=3, and thecytotoxin is crizotinib (1).

TABLE 7 Kinase inhibitors and analogues. Parent Toxin Structure toxinname 1

Crizotinib T1a

entrecitinib T3a

pelitinib T4a

lapatinib T8a

bosutinib T10a

imatinib T14a

vandetanib T15a

bosutinib 18a

sunutinib T19a

ponatinib T21a

masitinib T27a

nintedanib T29a

ceritinib T31a

palbociclib T32a

osimertinib T33a

Olmutinib

The procedure for the synthesis of target OS002 is illustrated in FIG. 6and described below.

Preparation of tert-butyl(2-(((4-nitrophenoxy)carboxyl)oxy)ethyl)carbamate (4). To a mixture oftert-butyl (2-hydroxyethyl)carbamate 2 (1.2 g, 7.45 mmol) andtriethylamine (2.58 mL, 18.6 mmol) in CH₂Cl₂ (80 mL) was addedp-nitrophenyl chloroformate 3 (1.5 g, 7.45 mmol) portion wise at 0° C.The resultant mixture was warmed to room temperature and stirred for 5h. The mixture was quenched with 10 mL saturate NH₄Cl solution,extracted with CH₂Cl₂ (2×50 mL). The combined organic layers were washedwith brine (20 mL), dried over Na₂SO₄, and evaporated. The crude residuewas purified by normal phase chromatography with 0-100% EtOAc-hexanes togive 4 (1.92 g, 79%). ¹H-NMR (300 MHz, CDCl₃): δ 8.28 (d, 2H), 7.39 (d,2H), 4.85-4.95 (m, 1H), 4.33 (t, 2H), 3.0-3.51 (m, 2H), 1.45 (s, 9H).

Preparation of 2-((tert-butoxycarboxyl)amino)ethyl(S)-4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate(5). To a mixture of crizotinib hydrochloride 1 (100 mg, 0.2 mmol) and 4(136 mg, 0.41 mmol) in CH₂Cl₂ (50 mL) was added DIPEA (0.4 mL g, 0.67mmol) at room temperature. The resultant mixture was stirred for 16 h.The mixture was washed with water (2×20 mL). The organic layer waswashed with brine (20 mL), dried over Na₂SO₄, and evaporated. The cruderesidue was purified by normal phase chromatography with 0-10%MeOH—CH₂Cl₂ to give 5 (130 mg, 99%). ¹H-NMR (300 MHz, CDCl₃): δ 7.75 (d,1H), 7.55 (s, 1H), 7.47 (s, 1H), 7.25-7.27 (m, 1H), 7.01-7.08 (m, 1H),6.85 (d, 1H), 6.05-6.1 (m, 1H), 475-4.85 (m, 3H), 4.1-4.35 (m, 5H),3.39-3.45 (m, 2H), 2.9-3.0 (m, 2H), 2.1-2.19 (m, 2H), 1.9-1.98 (m, 2H),1.86 (d, 3H), 12.1-2.19 (m, 2H), 1.9-1.98 (m, 2H), 1.86 (d, 3H), 1.43(s, 9H). MS m/z=637.2 [M]⁺. t_(R)=5.16.

Preparation of 2-aminoethyl(S)-4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate. HCl (6). To amixture of 5 (125 mg, 0.19 mmol) in THF (10 mL) was added HCl (4 mL, 4Nin dioxane) at room temperature. The resultant mixture was stirred for16 h. The solvents were evaporated and the resultant residue wastriturated with diethyl ether. The solid was filtered, and dried to give6 (100 mg, 94%). This was used in next step without furtherpurification. ¹H-NMR (300 MHz, DMSO-d₆): δ 8.03-8.13 (m, 3H), 7.71-7.85(m, 3H), 7.53-7.63 (m, 2H), 7.42-7.52 (m, 1H), 7.12 (s, 1H), 6.21-6.31(m, 1H), 4.25-4.45 (m, 1H), 4.05-4.19 (m, 4H), 2.9-3.07 (m, 4H),1.91-2.05 (m, 2H), 1.80-1.89 (m, 5H). MS m/z=537.2 [M]⁺. HPLCPurity=98%, t_(R)=4.72.

Preparation of4-(10,15,20-tris(4-(methoxycarbonyl)phenyl)porphyrin-5-yl)benzoic acid(8). A mixture of tetramethyl porphyrin derivative 7 (1.0 g, 1.18 mmol)and Me₃SnOH (0.43 g, 2.36 mmol) in 1,2-dichloroethane (10 mL) wasreacted in a microwave reactor at 150° C. for 1 h. The solvents wereevaporated and the residue absorbed in silica for purification. Thesilica absorbed crude compound was purified by normal phasechromatography with 0-10% MeOH—CH₂Cl₂ (0.1% CH₃COOH) to get the desiredcompound 8 (275 mg, 28%). ¹H-NMR (300 MHz, DMSO-d₆): δ 17.0 (s, 1H),13.2 (brs, 1H), 8.81-8.82 (m, 8H), 8.35-8.37 (m, 16H), 4.03 (s, 9H).

Preparation of trimethyl4,4′,4″-(20-(4-((2-((4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxyl)oxy)ethyl)carbamoyl) phenyl)porphyrin-5,10,15-triyl)(S)-tribenzoate (TARGETOS002). To a mixture of 6 (100 mg, 0.165 mmol), 8 (143 mg, 0.165 mmol)and PyBOP (136 mg, 0.248 mmol) in NMP (10 mL) was added DIPEA (0.2 mL,0.99 mmol). The resultant mixture was stirred at ambient temperatureovernight. The mixture was quenched with water (2 mL), extracted withCH₂Cl₂ (2×20 mL). The combined organic layers were washed with water,dried over Na₂SO₄, and evaporated. The crude compound was purified bynormal phase chromatography with 0-10% MeOH—CH₂Cl₂ to get the desiredtarget compound OS002 (132 mg, 56%) as purple solid. ¹H-NMR (300 MHz,CDCl₃): δ 12.17 (s, 1H), 8.81 (d, 8H), 8.45 (d, 6H), 8.18-8.29 (m, 10H),7.72 (s, 1H), 7.43-7.53 (m, 3H), 7.14-7.18 (m, 1H), 6.93 (t, 1H), 6.79(s, 1H), 5.9-6.1 (m, 1H), 4.72 (s, 2H), 4.53 (t, 1H), 4.2-4.48 (m 3H),4.10 (s, 9H), 3.89-3.95 (m, 2H), 2.9-3.15 (m, 2H), 2.13-2.23 (m, 2H),1.9-2.19 (m, 2H), 1.77 (d, 3H), Purity by HPLC: >96%, t_(R)=7.69.

HPLC Condition.

Agilent Tech 1200 series HPLC System equipped with Variable WavelengthDetector and Mass Spectrometer.

Column: Agela, Durashell C18, 3.0 μm, 4.60×50 mm.

Mobile Phase: A (ACN with 0.1% TFA)Mobile Phase: D (H₂O with 0.1% TFA)

Gradient

Time A (ACN with 0.1% TFA) D (H₂O with 0.1% TFA) 0 5 95 5.75 95 5 8 95 59 5 95

Detection: UV at 254 nm

In the synthetic scheme for OS002, the condensation of aryl carboxylate8 with the primary amine 6 using the amidation reagent PyBOP to affordOS002 is illustrated. In an embodiment, aryl carboxylate 8 may beexchanged for carboxylate P1, where the carboxylic acid substituent(COOH) occupies ortho, meta or para, that is, positions 2, 3 or 4, andwherein the three substituents L, M and N may independently occupy theortho, meta or para (positions 2, 3 or 4) on their respective aromaticrings.

The substituents L, M and N are selected from the group consisting of:

-   -   a) H;    -   b) carboxyaryl esters and acids (COOR) wherein the R may be H,        lower straight chain or branched alkyl, cycloalkyl,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, hetero-substituted cycloalkyl and        additionally, sugars. In addition, R may include aryl and        heteroaryl substituents;    -   c) Carboxamides (CONR1R2), wherein the R1 and R2 groups may        individually be selected from H, be lower straight chain or        branched alkyl, cycloalkyl, hydroxy-substituted alkyl,        polyethers (lower PEG), amino-substituted alkyl,        hetero-substituted cycloalkyl. In addition, R may include aryl        and heteroaryl substituents;    -   d) Aminoaryl groups (NR1R2) wherein R1 or R2 may individually be        selected from the group lower alkyl, branched lower alkyl,        cycloalkyl, or hetero-substituted alkyl or hetero-substituted        cycloalkyl. R may also be an acyl group such as C(O)R, where R        may include lower alkyl, hydroxy-substituted alkyl, polyethers        (lower PEG), amino-substituted alkyl, cycloalkyl. In addition, R        may include aryl and heteroaryl substituents;    -   e) Sulfur containing functional groups that may include thiols,        sulfonic acids and corresponding amides thereof. The amides may        be further defined as containing a nitrogen moiety NHR, with the        N connected by a single bond to the sulfur atom, and where R is        lower alkyl, cycloalkyl, or hetero-substituted alkyl or        hetero-substituted cycloalkyl. R may also be an acyl group such        as C(O)R, where R may include lower alkyl, hydroxy-substituted        alkyl, polyethers (lower PEG), amino-substituted alkyl,        cycloalkyl. In addition, R may include aryl and heteroaryl        substituents;    -   f) Lower straight chain or branched alkyl groups, cycloalkyl        groups, hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl; and    -   g) Oxygen groups such as hydroxy and substituted hydroxyaryl (OH        or OR), where R may be chosen from the group including lower        straight chain or branched alkyl groups, cycloalkyl groups,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl. In addition, R may include acyl (C(O)R),        carbamoyloxy (C(O)NR1R2), wherein R1 and R2 are lower alkyl,        aryl and heteroaryl substituents.

In a further embodiment, the carboxylate substituent (COOH) occupies thepara (4-position) on its aromatic ring and further, the substituents oneach of the other three aromatic rings are identical, that is L=M=N.Moreover, the substituents L, M and N are situated such that theirpositions on each aromatic ring are identical; for example, where eachsubstituent occupies the meta position on its respective aromatic ring.A preferred embodiment is where the substituents L, M and N are allcarboxylate (COOH) or carboxymethyl (COOMe) and are situated at the paraposition (i.e., 4-position) of their respective aromatic rings.

As shown in FIG. 6 , the starting material tert-butyl(2-hydroxyethyl)carbamate (2) is utilized as a key building block forthe linker moiety that connects the porphyrin 8 to crizotinib by way ofa carbamate bond to the nitrogen of crizotinib and an amide bond to thecarboxylate of 8. In one embodiment, the starting material 2 is replacedby a protected aminoalcohol L5 or L6, wherein the group P represents anamine protecting group that may include carbamates (e.g., BOC, FMOC,Alloc, CBZ), amides (e.g., acetamide, dichloroacetamide) or otherprotecting groups containing a bond between the nitrogen of the amineand either carbon or silicon of the protecting group (e.g., benzyl,tert-butyl, triisopropylsilyl). In L5 or L6, n=1-12. In a preferredembodiment, the aminoalcohol L5 is chosen such that P is BOC and n=1.

FIG. 6 illustrates the preparation of key intermediate 6, whereincrizotinib is functionalized at its secondary amine by a carbamoyl aminemoiety. In an embodiment, the toxin, kinase inhibitor crizotinib, of (1)is replaced with a kinase inhibitor chosen from the set Tna (Table 7). Xrepresents the amide bond to L5 or L6. In a preferred embodiment, P1 isthe porphyrin where L, M and N are all carboxylic acid or carboxymethylgroups positioned para to the porphyrin rings, and where the carboxylicacid group forming the amide bond to the linker, is positioned pararelative to the porphyrin ring. Moreover, L5 is used, where P=H and n=1and the kinase inhibitor is crizotinib.

The procedure for the synthesis of target OS009 is illustrated in FIG.15 and described below.

Preparation of(S)-4,4′,4″-(20-(4-((2-((4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carbonyl)oxy)ethyl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoic acid (2). To a mixture of OS002 (35mg, 2.03 mmol) in THF:MeOH:H₂O (1.5 mL, 3:1:1) was added LiOH. H₂O (7mg, 0.166 mmol). The resultant mixture was stirred for 64 h at roomtemperature. The solvents were evaporated and the pH of the reactionmixture was adjusted to pH 6-7, by addition of 0.1 M aqueous HClsolution, and lyophilized. An additional batch was similarly prepared on25 mg scale following this procedure. The combined batches were purifiedby preparative HPLC using acetonitrile-water (0.1% TFA) as solventsystem to give the desired compound 2 (17.5 mg, 30%) as green solid. MS(ESI) m/z=1309.3 [M]⁺. Purity by HPLC (ELSD): >97%, t_(R)=6.0.

HPLC Condition.

Agilent Tech 1100 series HPLC System equipped with Variable WavelengthDetector and ELSD Detector

Column: Agela, Durashell C18, 3.0 μm, 4.60×50 mm.

Mobile Phase: A (ACN with 0.1% TFA)Mobile Phase: D (H₂O with 0.1% TFA)

The procedure for the synthesis of target OS0024 is illustrated in FIG.7 and described below.

STEP 1: Synthesis of ethyl5-(3-(10,15,20-tris(3-hydroxyphenyl)porphyrin-5-yl)phenoxy) pentanoate)(2). A mixture of meso-tetrakis(3-hydroxyphenyl)porphyrin 1 (1.25 g,1.84 mmol) and K₂CO₃ (0.5 g) in DMF (30 mL) was stirred under nitrogenat room temperature for 30 min. Ethyl 5-bromopentanoate (1.15 g, 5.5mmol, 3 eq.) was added. The mixture was stirred at room temperatureovernight. The reaction mixture was diluted with DCM, washed with water,satd.NaHCO₃ (aq), water and brine, dried over Na₂SO₄, and evaporated.The crude residue was purified with two successive columnchromatographies to give 2 (0.42 g, 28%). ¹H-NMR (300 MHz, DMSO-d₆): δ12.0 (s, 1H), 9.90 (s, 3H), 8.88 (s, 8H), 7.66-7.79 (m, 3H), 7.52-7.65(m, 9H), 7.36-7.7.42 (m, 1H), 7.18-7.26 (m, 3H), 4.12-4.20 (m, 2H), 4.05(q, 2H), 2.34-2.40 (m, 2H), 1.65-1.85 (m, 4H), 1.12 (t, 3H).

STEP 2: Synthesis of5-(3-(10,15,20-tris(3-hydroxyphenyl)porphyrin-5-yl)phenoxy) pentanoicacid (3). To a solution of 2 (157 mg, 0.19 mmol) in THF (45 mL) wasadded a solution of LiOH—H2O (0.15 g) in water (30 mL) at roomtemperature. The mixture was stirred at room temperature overnight.Evaporated THF, diluted with sat. NH₄Cl, extracted with DCM, washed withbrine, and evaporated. It was used for next step reaction withoutfurther purification. 3 (0.14 g, 92%); 1H-NMR (300 MHz, DMSO-d6): δ 12.0(s, 2H), 9.90 (s, 3H), 8.89 (s, 8H), 7.66-7.82 (m, 3H), 7.54-7.65 (m,9H), 7.36-7.7.42 (m, 1H), 7.20-7.26 (m, 3H), 4.14-4.22 (m, 2H),2.26-2.36 (m, 2H), 1.65-1.85 (m, 4H).

STEP 3: SynthesisN-((2S,3S,4R,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)-5-(3-(10,15,20-tris(3-hydroxyphenyl)porphyrin-5-yl)phenoxy)pentanamide (OS0024). To a solution of 3 (53 mg, 0.068 mmol) in DMF (5.5mL) was added HATU (56 mg, 0.147 mmol, 2.16 eq.) and DIPEA (75 mL).Stirred at room temperature for 5 min. followed by addition ofdoxorubicin-HCl (35 mg, 0.06 mmol, 0.9 eq.). The mixture was stirred atroom temperature overnight. DMF was evaporated and the residue waspurified by a normal phase column chromatography followed by apreparatory-HPLC using ACN/Water with TFA as eluent to afford thedesired target compound OS0024 (5 mg). MS(ES) [M+1]+=1304; HPLC purity:90% (UV400) and ELSD (96%).

HPLC Condition

Agilent Tech 1200 series HPLC System equipped with Variable WavelengthDetector and Mass Spectrometer and ELSD Detector

Column: Agela, Durashell C18, 3.0 μm, 4.60×50 mm.

Mobile Phase: A (ACN with 0.1% TFA)Mobile Phase: D (H₂O with 0.1% TFA)

Gradient

Time A (ACN with 0.1% TFA) D (H₂O with 0.1% TFA) 0 5 95 5.75 95 5 8 95 59 5 95Detection: UV at 400 nm and evaporative light scattering detector.

FIG. 7 illustrates the conjugation of porphyrin 1 with doxorubicin via acovalent linker whose carbons derive from the bifunctional linkerstarting material ethyl 5-bromopentanoate. In an embodiment, porphyrin 1could be substituted by porphyrin P4,

wherein the hydroxy substituent could be situated at the ortho, meta orpara positions (positions 2, 3 or 4) of the aromatic ring and, moreover,where the substituents L, M and N, may be independently occupy theortho, meta or para, that is 2-, 3- or 4-positions, on their respectivearomatic rings. The substituents L, M ad N are selected from the setconsisting of:

-   -   a) H;    -   b) carboxyaryl esters and acids (COOR) where the R may be H,        lower straight chain or branched alkyl, cycloalkyl,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, hetero-substituted cycloalkyl and        additionally, sugars. In addition, R may include aryl and        heteroaryl substituents;    -   c) Carboxamides (CONR1R2), where wherein the R1 and R2 groups        may individually be H, be lower straight chain or branched        alkyl, cycloalkyl, hydroxy-substituted alkyl, polyethers (lower        PEG), amino-substituted alkyl, hetero-substituted cycloalkyl. In        addition, R may include acyl (C(O)R1), carbamoyloxy (C(O)NR1R2),        aryl and heteroaryl substituents;    -   d) Aminoaryl groups (NR1R2) where R1 or R2 may individually be        selected from the group lower alkyl, branched lower alkyl,        cycloalkyl, or hetero-substituted alkyl or hetero-substituted        cycloalkyl. R may also be an acyl group such as C(O)R, where R        may include lower alkyl, hydroxy-substituted alkyl, polyethers        (lower PEG), amino-substituted alkyl, cycloalkyl. In addition, R        may include aryl and heteroaryl substituents;    -   e) Sulfur containing functional groups that may include thiols,        sulfonic acids and corresponding amides thereof. The amides may        be further defined as containing a nitrogen moiety NHR, with the        N connected by a single bond to the sulfur atom, and where R is        lower alkyl, cycloalkyl, or hetero-substituted alkyl or        hetero-substituted cycloalkyl. R may also be an acyl group such        as C(O)R, where R may include lower alkyl, hydroxy-substituted        alkyl, polyethers (lower PEG), amino-substituted alkyl,        cycloalkyl. In addition, R may include aryl and heteroaryl        substituents;    -   f) Lower straight chain or branched alkyl groups, cycloalkyl        groups, hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl;    -   g) Oxygen groups such as hydroxy and substituted hydroxyaryl (OH        or OR), where R may be chosen from the group including lower        straight chain or branched alkyl groups, cycloalkyl groups,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl. In addition, R may include acyl, aryl and heteroaryl        substituents; and    -   h) Cyano or halogens (F, Cl, Br, I).

In a further embodiment, the aromatic hydroxy substituent (OH) occupiesthe meta (3 position) on its aromatic ring and, further, thesubstituents on each of the other three aromatic rings are identical,that is L=M=N. Moreover, the substituents L, M and N are situated suchthat their positions on each aromatic ring are identical; for example,where each substituent occupies the meta position on its respectivearomatic ring. In a preferred embodiment, the substituents L, M and Nare all hydroxyl (OH) and are situated at the meta (3 position) of theirrespective aromatic rings.

As shown in FIG. 7 , the phenol group of the porphyrin is alkylated viaan S_(N)2 reaction under basic conditions with ethyl 5-bromopentanoateto give intermediate 2. In an embodiment, ethyl 5-bromopentanoate may bereplaced by L1 or L2. In particular, n is selected from 1-12 for L1 andL2. X is a leaving group suitable for an S_(N)2 reaction with a phenolicoxygen atom to form a phenolic ether (e.g., 2 in FIG. 7 ). In thiscontext, the leaving group X is either a halogen leaving groups (Cl, Br,I) or a variety of activated sulfonyl esters such as mesylates,tosylates or triflates. Moreover, Y on L1 or L2 may be a variety ofcarboxylate esters (OR), wherein R may be chosen to be H, lower straightchain or branched alkyl, cycloalkyl, aryl or heteroaryl. In a preferredembodiment, X is bromo and Y is ethoxy.

Attachment to the doxorubicin occurs via the saponification of the estergroup of intermediate 2 to the corresponding carboxylic acid (3) andamide bond formation between the carboxylic acid and the amine on theaminosugar moiety of doxorubicin using the peptide coupling reagentHATU, to afford OS0024. The toxin conjugated in FIG. 7 is doxorubicin,but could more generally be chosen from the anthracycline antibioticspossessing an aminosugar moiety capable of forming an amide bond asillustrated in Table 6, wherein doxorubicin is denoted as T1b andexamples of analogues are illustrated as T2b-T4b. In a preferredembodiment, P4 is selected such that all the substituents (L=M=N) arehydroxyl groups positioned meta to the porphyrin ring and wherein L1 isselected such that n=3, X is bromo and Y is ethoxy. Moreover, in thispreferred embodiment, the toxin is selected as doxorubicin (T1b).

The procedure for synthesis of target OS007 is illustrated in FIG. 8 anddescribed below.

Preparation of4-(10,15,20-tris(4-(methoxycarbonyl)phenyl)porphyrin-5-yl)benzoic acid(2). A mixture of tetrakis(4-carbomethoxyphenyl)porphyrin (1) (1.0 g,1.18 mmol) and Me₃SnOH (0.43 g, 2.36 mmol) in 1,2-dichloroethane (10 mL)was reacted in a microwave reactor at 150° C. for 1 h. The solvents wereevaporated and absorbed on silica gel for purification. The silicaabsorbed crude compound was purified by normal phase chromatography with0-10% MeOH—CH₂Cl₂ (0.1% CH₃COOH) to get the desired compound 2 (275 mg,28%). ¹H-NMR (300 MHz, DMSO-d₆): δ 17.0 (s, 1H), 13.2 (brs, 1H),8.81-8.82 (m, 8H), 8.35-8.37 (m, 16H), 4.03 (s, 9H).

Preparation of6-(4-(10,15,20-tris(4-(methoxycarbonyl)phenyl)porphyrin-5-yl)benzamido)hexanoic acid (4). A mixture of 2 (300 mg, 0.36 mmol), PyBOP (374 mg,0.72 mmol) and DIPEA (0.48 mL, 2.8 mmol) in NMP (15 mL) was stirred for10 min at room temperature. To the above mixture was added6-aminohexanoic acid (3, 188 mg, 1.44 mmol) and the resultant mixturewas stirred at room temperature for 48 h. The mixture was diluted withCH₂Cl₂, washed with water (3×50 mL). The organic layer was dried overNa₂SO₄, and evaporated. The residue was purified by normal phasechromatography with 0-10% MeOH—CH₂Cl₂ to get the desired compound 4 (89mg, 26%). ¹H-NMR (300 MHz, CDCl₃): δ 12.17 (s, 1H), 8.80 (s, 8H),8.14-8.44 (m, 16H), 6.39 (brt, 1H), 3.51-3.62 (m, 2H), 2.40-2.45 (m,2H), 1.45-1.79 (m, 6H).

Preparation of trimethyl4,4′,4″-(20-(44(6-(42S,3S,4S,6R)-3-hydroxy-2-methyl-6-(41S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)amino)-6-oxohexyl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoate(OS007). To a mixture of 4 (20 mg, 0.021 mmol), HATU (19.2 mg, 0.051mmol) and DIPEA (30 μL, 0.168 mmol) in DMF (2.5 mL) was stirred for 10min at room temperature. To the above mixture was added doxorubicin.HCl(5, 13.5 mg, 0.023 mmol) and the resultant mixture was stirred at roomtemperature for 16 h. The mixture was diluted with CH₂Cl₂, washed withwater (3×10 mL). The organic layer was dried over Na₂SO₄, andevaporated. The residue was purified by normal phase chromatography with0-10% MeOH—CH₂Cl₂ to afford the desired compound OS007 (21 mg, 68%). %).¹H-NMR (300 MHz, DMSO-d₆): δ 12.0 (s, 1H), 9.90 (s, 3H), 8.88 (s, 8H),7.66-7.79 (m, 3H), 7.52-7.65 (m, 9H), 7.36-7.7.42 (m, 1H), 7.18-7.26 (m,3H), 4.12-4.20 (m, 2H), 4.05 (q, 2H), 2.34-2.40 (m, 2H), 1.65-1.85 (m,4H), 1.12 (t, 3H).

In FIG. 8 , the porphyrin carboxylate 2 is condensed with6-aminohexanoic acid (3) using PyBOP to afford an amide (4). In anembodiment, amino acid (3) is replaced with amino acids L3 or L4,wherein the group P represents H. The value of n in L3 and L4 isselected from 1-12 and Y is OH.

The anthracycline toxin illustrated in FIG. 8 is doxorubicin. However,in an embodiment, the anthracycline may be selected from the groupT1b-T4b, where T1b is doxorubicin (Table 6).

FIG. 8 further describes the condensation of the amine in intermediate 2with the carboxylic acid group of 4 using ByPOP to afford the conjugateOS007. Porphyrin 4 is in turn derived from porphyrins 2, which may bereplaced by P1, wherein the carboxylic acid substituent (COOH) occupiesortho, meta or para positions, that is, positions 2, 3 or 4, and whereinthe three substituents L, M and N may independently occupy the ortho,meta or para (positions 2, 3 or 4) on their respective aromatic rings.The substituents L, M ad N are selected from the set consisting of:

-   -   a) H;    -   b) carboxyaryl esters and acids (COOR) where the R may be H,        lower straight chain or branched alkyl, cycloalkyl,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, hetero-substituted cycloalkyl and        additionally, sugars. In addition, R may include aryl and        heteroaryl substituents;    -   c) Carboxamides (CONR1R2), where wherein the R1 and R2 groups        may individually be H, be lower straight chain or branched        alkyl, cycloalkyl, hydroxy-substituted alkyl, polyethers (lower        PEG), amino-substituted alkyl, hetero-substituted cycloalkyl. In        addition, R may include aryl and heteroaryl substituents;    -   d) Aminoaryl groups (NR1R2) where R1 or R2 may individually be        selected from the group lower alkyl, branched lower alkyl,        cycloalkyl, or hetero-substituted alkyl or hetero-substituted        cycloalkyl. R may also be an acyl group such as C(O)R, where R        may include lower alkyl, hydroxy-substituted alkyl, polyethers        (lower PEG), amino-substituted alkyl, cycloalkyl. In addition, R        may include aryl and heteroaryl substituents;    -   e) Sulfur containing functional groups that may include thiols,        sulfonic acids and corresponding amides thereof. The amides may        be further defined as containing a nitrogen moiety NHR, with the        N connected by a single bond to the sulfur atom, and where R is        lower alkyl, cycloalkyl, or hetero-substituted alkyl or        hetero-substituted cycloalkyl. R may also be an acyl group such        as C(O)R, where R may include lower alkyl, hydroxy-substituted        alkyl, polyethers (lower PEG), amino-substituted alkyl,        cycloalkyl. In addition, R may include aryl and heteroaryl        substituents;    -   f) Lower straight chain or branched alkyl groups, cycloalkyl        groups, hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl;    -   g) Oxygen groups such as hydroxy and substituted hydroxyaryl (OH        or OR), where R may be chosen from the group including lower        straight chain or branched alkyl groups, cycloalkyl groups,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl. In addition, R may include acyl (C(O)R),        carbamoyloxy (C(O)NR1R2), aryl and heteroaryl substituents; and    -   h) Cyano or halogens (F, Cl, Br, I).

In a further embodiment, the carboxylate substituent (COOH) occupies thepara (4-position) on its aromatic ring and further, the substituents oneach of the other three aromatic rings are identical, that is L=M=N.Moreover, the substituents L, M and N are situated such that theirpositions on each aromatic ring are identical; for example, where eachsubstituent occupies the meta position on its respective aromatic ring.A preferred embodiment is where the substituents L, M and N are allcarboxylate (COOH) or carboxymethyl (COOMe) and are situated at the paraposition (i.e., 4-position) of their respective aromatic rings.

The procedure for synthesis of target OS0030 is illustrated in FIG. 9and described below.

Preparation of4-(10,15,20-tris(4-(methoxycarbonyl)phenyl)porphyrin-5-yl)benzoic acid(2). A mixture of 1 (1.0 g, 1.18 mmol) and Me₃SnOH (0.43 g, 2.36 mmol)in 1,2-dichloroethane (10 mL) was reacted in a microwave reactor at 150°C. for 1 h. The solvents were evaporated and absorbed on silica gel forpurification. The silica-absorbed crude compound was purified by normalphase chromatography with 0-10% MeOH—CH₂Cl₂ (0.1% CH₃COOH) to get thedesired compound 2 (275 mg, 28%). ¹H-NMR (300 MHz, DMSO-d₆): δ 17.0 (s,1H), 13.2 (brs, 1H), 8.81-8.82 (m, 8H), 8.35-8.37 (m, 16H), 4.03 (s,9H).

Preparation of trimethyl4,4′,4″-(20-(4-((2-hydroxyethyl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoate(4). To a mixture of 2 (20 mg, 0.024 mmol), ethanol amine 3 (5.8 mg,0.096 mmol) and PyBOP (19 mg, 0.036 mmol) in NMP (1 mL) was added DIPEA(20 μL, 0.12 mmol). The resultant mixture was stirred at ambienttemperature overnight. The mixture was quenched with water (1 mL),extracted with CH₂Cl₂ (2×5 mL). The combined organic layers were washedwith water, dried over Na₂SO₄, and evaporated. The crude compound waspurified by normal phase chromatography with 0-10% MeOH—CH₂Cl₂ to getthe desired compound 4 (14 mg, 67%) as purple solid. ¹H-NMR (300 MHz,CDCl₃): δ 12.09 (s, 1H), 8.80 (s, 8H), 8.26-8.45 (m, 16H), 6.92 (brs,1H), 4.10 (s, 9H), 3.98-4.01 (m, 2H), 3.80-3.84 (m, 2H).

Preparation of trimethyl4,4′,4″-(20-(4-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoate (6). To a mixtureof 4 (12 mg, 0.0137 mmol) and 5 (4 mg, 0.049 mmol) in CH₂Cl₂:DMF (8:2, 1mL) was added DIPEA (12 μL g, 0.068 mmol) at room temperature. Theresultant mixture was stirred for 4 h. The mixture was diluted with DCM(2 mL) and washed with water (2×10 mL), dried over Na₂SO₄, andevaporated. The crude residue was purified by normal phasechromatography with 0-10% MeOH—CH₂Cl₂ to obtain 6 (8 mg, 57%). ¹H-NMR(300 MHz, CDCl₃): δ 12.17 (s, 1H), 8.81 (s, 8H), 8.16-8.43 (m, 18H),7.42 (d, 2H), 6.84 (brt, 1H), 4.62-4.64 (m, 2H), 3.9-4.11 (m, 11H).

Preparation of trimethyl4,4′,4″-(20-(4-((2-((((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)ethyl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoate (OS0030). To a mixture of 6(8 mg, 0.0076 mmol) and doxorubicin.HCl 7 (3.7 mg, 0.0064 mmol) in DMF(0.8 mL) was added DIPEA (10 μL g, 0.061 mmol) at room temperature. Theresultant mixture was stirred for 4 h. The mixture was diluted withCH₂Cl₂ (5 mL), The mixture was washed with water (2×5 mL), dried overNa₂SO₄, and evaporated. The crude residue was purified by normal phasechromatography with 0-10% MeOH—CH₂Cl₂ to give OS0030 (7 mg, 77%). ¹H-NMR(300 MHz, DMSO-d₆): δ 12.0 (s, 1H), 9.90 (s, 3H), 8.88 (s, 8H),7.66-7.79 (m, 3H), 7.52-7.65 (m, 9H), 7.36-7.7.42 (m, 1H), 7.18-7.26 (m,3H), 4.12-4.20 (m, 2H), 4.05 (q, 2H), 2.34-2.40 (m, 2H), 1.65-1.85 (m,4H), 1.12 (t, 3H).

In FIG. 9 , the hydroxycarboxamide (4) is formed from the condensationof ethanolamine (3) with porphyrin carboxylate (2). In an embodiment,ethanolamine (3) may be replaced by aminoalcohols L5 or L6, wherein thegroup P represents H and n=1-12. The toxin illustrated in FIG. 9 isdoxorubicin. However, in an embodiment, the anthracycline may beselected from the group T1b-T4b, where T1b is doxorubicin (Table 6). Thecarboxyaryl porphyrin (2) illustrated in FIG. 9 may, in an embodiment,also may be replaced by P1, where the carboxylic acid substituent (COOH)occupies ortho, meta or para, that is, positions 2, 3 or 4, and whereinthe three substituents L, M and N may independently occupy the ortho,meta or para (positions 2, 3 or 4) on their respective aromatic rings.The substituents L, M ad N are selected from the set consisting of:

-   -   a) H;    -   b) carboxyaryl esters and acids (COOR) where the R may be H,        lower straight chain or branched alkyl, cycloalkyl,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, hetero-substituted cycloalkyl and        additionally, sugars. In addition, R may include aryl and        heteroaryl substituents;    -   c) Carboxamides (CONR1R2), where wherein the R1 and R2 groups        may individually be H, be lower straight chain or branched        alkyl, cycloalkyl, hydroxy-substituted alkyl, polyethers (lower        PEG), amino-substituted alkyl, hetero-substituted cycloalkyl. In        addition, R may include aryl and heteroaryl substituents;    -   d) Aminoaryl groups (NR1R2) where R1 or R2 may individually be        selected from the group lower alkyl, branched lower alkyl,        cycloalkyl, or hetero-substituted alkyl or hetero-substituted        cycloalkyl. R may also be an acyl group such as C(O)R, where R        may include lower alkyl, hydroxy-substituted alkyl, polyethers        (lower PEG), amino-substituted alkyl, cycloalkyl. In addition, R        may include aryl and heteroaryl substituents;    -   e) Sulfur containing functional groups that may include thiols,        sulfonic acids and corresponding amides thereof. The amides may        be further defined as containing a nitrogen moiety NHR, with the        N connected by a single bond to the sulfur atom, and where R is        lower alkyl, cycloalkyl, or hetero-substituted alkyl or        hetero-substituted cycloalkyl. R may also be an acyl group such        as C(O)R, where R may include lower alkyl, hydroxy-substituted        alkyl, polyethers (lower PEG), amino-substituted alkyl,        cycloalkyl. In addition, R may include aryl and heteroaryl        substituents;    -   f) Lower straight chain or branched alkyl groups, cycloalkyl        groups, hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl;    -   g) Oxygen groups such as hydroxy and substituted hydroxyaryl (OH        or OR), where R may be chosen from the group including lower        straight chain or branched alkyl groups, cycloalkyl groups,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl. In addition, R may include acyl (C(O)R),        carbamoyloxy (C(O)NR1R2), wherein R1 and R2 may be H or lower        alkyl, aryl and heteroaryl substituents.

In a preferred embodiment, the porphyrin is selected to be P1, whereinthe carboxylic acid group is in the para position relative to theporphyrin ring, L, M and N are all carboxylic acid or carboxymethylester groups and are all in the para position relative to the porphyrinring. Moreover, in this preferred embodiment, L5 is selected such thatn=1 and P=H.

The procedure for synthesis of target OS0035 is illustrated in FIG. 10and described below.

Preparation of4-(10,15,20-tris(4-(methoxycarbonyl)phenyl)porphyrin-5-yl)benzoic acid(2). A mixture of 1 (1.0 g, 1.18 mmol) and Me₃SnOH (0.43 g, 2.36 mmol)in 1,2-dichloroethane (10 mL) was reacted in a microwave reactor at 150°C. for 1 h. The solvents were evaporated and absorbed on silica gel forpurification. The silica absorbed crude compound was purified by normalphase chromatography with 0-10% MeOH—CH₂Cl₂ (0.1% CH₃COOH) to get thedesired compound 2 (275 mg, 28%). ¹H-NMR (300 MHz, DMSO-d₆): δ 17.0 (s,1H), 13.2 (brs, 1H), 8.81-8.82 (m, 8H), 8.35-8.37 (m, 16H), 4.03 (s,9H).

Preparation of6-(4-(10,15,20-tris(4-(methoxycarbonyl)phenyl)porphyrin-5-yl)benzamido)hexanoic acid (4). A mixture of 2 (300 mg, 0.36 mmol), PyBOP (374 mg,0.72 mmol) and DIPEA (0.48 mL, 2.8 mmol) in NMP (15 mL) was stirred for10 min. at room temperature. To the above mixture was added6-aminohexanoic acid (3, 188 mg, 1.44 mmol) and the resultant mixturewas stirred at room temperature for 48 h. The mixture was diluted withCH₂Cl₂, washed with water (3×50 mL). The organic layer was dried overNa₂SO₄, and evaporated. The residue was purified by normal phasechromatography with 0-10% MeOH—CH₂Cl₂ to obtain the desired compound 4(89 mg, 26%). ¹H-NMR (300 MHz, CDCl₃): δ 12.17 (s, 1H), 8.80 (s, 8H),8.14-8.44 (m, 16H), 6.39 (brt, 1H), 3.51-3.62 (m, 2H), 2.40-2.45 (m,2H), 1.45-1.79 (m, 6H).

Preparation trimethyl4,4′,4″-(20-(4-(((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-41R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2-oxa-5,8,11-triazaheptadecan-17-yl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoate(OS0035). To a mixture of 4 (21 mg, 0.022 mmol), HATU (20.2 mg, 0.053mmol) and DIPEA (31 μL, 0.177 mmol) in DMF (2 mL) was stirred for 10 minat room temperature. To the above mixture was added monomethylauristatin E (5, 17.5 mg, 0.024 mmol) followed by HOBt (3.3 mg, 0.024mmol) and the resultant mixture was stirred at room temperature for 16h. The mixture was diluted with CH₂Cl₂ and washed with water (3×10 mL).The organic layer was dried over Na₂SO₄, and evaporated. The residue waspurified by normal phase chromatography with 0-10% MeOH—CH₂Cl₂ to getthe desired target compound OS0035 (12 mg, 33%).

FIG. 10 illustrates the condensation of the secondary amine ofmonomethylauristatin E (5), with the carboxylate of intermediate 4,using peptide coupling reagent HATU, to afford OS0035. In an embodiment,the 5 may be replaced with auristatin-related peptides T5c, T9c and T10c(Table 8).

TABLE 8 Auristatin-related peptides

 T4c

 T5c

 T9c

T10c

In another embodiment, the amino acid 3 is replaced by a protected L3 orL4, where P=H and n=1-12 and wherein Y is OH.

In FIG. 10 , the porphyrin carboxylate (2) is condensed with the primaryamine 3 to form the amide bond affording the intermediate carboxylate 4,which in turn is condensed with MMAE (5) to afford OS0035. However, inanother embodiment, 2 may be replaced by P1, where the carboxylic acidsubstituent (COOH) occupies ortho, meta or para positon, that is,positions 2, 3 or 4, relative to the porphyrin ring and wherein thethree substituents L, M and N may independently occupy the ortho, metaor para (positions 2, 3 or 4) on their respective aromatic rings. Thesubstituents L, M ad N are selected from the set consisting of:

-   -   a) H;    -   b) carboxyaryl esters and acids (COOR) where the R may be H,        lower straight chain or branched alkyl, cycloalkyl,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, hetero-substituted cycloalkyl and        additionally, sugars. In addition, R may include aryl and        heteroaryl substituents;    -   c) Carboxamides (CONR1R2), where wherein the R1 and R2 groups        may individually be H, be lower straight chain or branched        alkyl, cycloalkyl, hydroxy-substituted alkyl, polyethers (lower        PEG), amino-substituted alkyl, hetero-substituted cycloalkyl. In        addition, R may include aryl and heteroaryl substituents;    -   d) Aminoaryl groups (NR1R2) where R1 or R2 may individually be        selected from the group lower alkyl, branched lower alkyl,        cycloalkyl, or hetero-substituted alkyl or hetero-substituted        cycloalkyl. R may also be an acyl group such as C(O)R, where R        may include lower alkyl, hydroxy-substituted alkyl, polyethers        (lower PEG), amino-substituted alkyl, cycloalkyl. In addition, R        may include aryl and heteroaryl substituents;    -   e) Sulfur containing functional groups that may include thiols,        sulfonic acids and corresponding amides thereof. The amides may        be further defined as containing a nitrogen moiety NHR, with the        N connected by a single bond to the sulfur atom, and where R is        lower alkyl, cycloalkyl, or hetero-substituted alkyl or        hetero-substituted cycloalkyl. R may also be an acyl group such        as C(O)R, where R may include lower alkyl, hydroxy-substituted        alkyl, polyethers (lower PEG), amino-substituted alkyl,        cycloalkyl. In addition, R may include aryl and heteroaryl        substituents;    -   f) Lower straight chain or branched alkyl groups, cycloalkyl        groups, hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl;    -   g) Oxygen groups such as hydroxy and substituted hydroxyaryl (OH        or OR), where R may be chosen from the group including lower        straight chain or branched alkyl groups, cycloalkyl groups,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl. In addition, R may include acyl (C(O)R),        carbamoyloxy (C(O)NR1R2), where R1 and R2 are lower alkyl, aryl        and heteroaryl substituents.

In a preferred embodiment, the porphyrin is selected to be P1, whereinthe carboxylic acid group is in the para position relative to theporphyrin ring, L, M and N are all carboxylic acid or carboxymethylester groups and are all in the para position relative to the porphyrinring. Moreover, in this preferred embodiment, L3 is selected such thatn=4 and P=H. In this embodiment, the toxin is T4c (Table 8).

The procedure for synthesis of target OS0032 is illustrated in FIG. 11and described below.

Preparation of4-(10,15,20-tris(4-(methoxycarbonyl)phenyl)porphyrin-5-yl)benzoic acid(2). A mixture of tetra methyl porphyrin ester (1) (1.0 g, 1.18 mmol)and Me3SnOH (0.43 g, 2.36 mmol) in 1,2-dichloroethane (10 mL) wasreacted in a microwave reactor at 150° C. for 1 h. The solvents wereevaporated and absorbed on silica gel for purification. The silicaabsorbed crude compound was purified by normal phase chromatography with0-10% MeOH—CH₂Cl₂ (0.1% CH₃COOH) to get the desired compound 2 (275 mg,28%). ¹H-NMR (300 MHz, DMSO-d6): δ 17.0 (s, 1H), 13.2 (br s, 1H),8.81-8.82 (m, 8H), 8.35-8.37 (m, 16H), 4.03 (s, 9H).

Preparation of trimethyl4,4′,4″-(20-(44(2-hydroxyethyl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoate(4). To a mixture of 2 (20 mg, 0.024 mmol), ethanol amine (3) (5.8 mg,0.096 mmol) and PyBOP (19 mg, 0.036 mmol) in NMP (1 mL) was added DIPEA(20 μL, 0.12 mmol). The resultant mixture was stirred at ambienttemperature overnight. The mixture was quenched with water (1 mL) andextracted with CH₂Cl₂ (2×5 mL). The combined organic layers were washedwith water, dried over Na₂SO₄, and evaporated. The crude compound waspurified by ISCO with 0-10% MeOH—CH₂Cl₂ to get the desired compound 4(14 mg, 67%) as purple solid. ¹H-NMR (300 MHz, CDCl₃): δ 12.09 (s, 1H),8.80 (s, 8H), 8.26-8.45 (m, 16H), 6.92 (brs, 1H), 4.10 (s, 9H),3.98-4.01 (m, 2H), 3.80-3.84 (m, 2H).

Preparation of trimethyl4,4′,4″-(20-(4-((2-(((4-nitrophenoxy)carbonyl)oxy) ethyl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoate (6). To a mixtureof 4 (12 mg, 0.0137 mmol) and p-nitrophenyl chloroformate 5 (4 mg, 0.049mmol) in CH₂Cl₂:DMF (8:2, 1 mL) was added DIPEA (12 μL g, 0.068 mmol) atroom temperature. The resultant mixture was stirred for 4 h. The mixturewas diluted with DCM (2 mL), washed with water (2×10 mL), dried overNa2SO4, and evaporated. The crude residue was purified by normal phasechromatography with 0-10% MeOH—CH₂Cl₂ to give 6 (8 mg, 57%). ¹H-NMR (300MHz, CDCl₃): δ 12.17 (s, 1H), 8.81 (s, 8H), 8.16-8.43 (m, 18H), 7.42 (d,2H), 6.84 (brt, 1H), 4.62-4.64 (m, 2H), 3.9-4.11 (m, 11H).

Preparation of trimethyl4,4′,4″-(20-(4-(((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-41R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,13-dioxa-5,8,11-triazapentadecan-15-yl)carbamoyl)phenyl)porphyrin-5,10,15-triyl)tribenzoate(OS0032). To a mixture of 6 (20 mg, 0.019 mmol) and monomethylauristatin E 7 (15.1 mg, 0.021 mmol) in DMF (0.8 mL) was added DIPEA (27μL g, 0.153 mmol) at room temperature. The resultant mixture was stirredfor 16 at ambient temperature. The mixture was diluted with CH₂Cl₂ (5mL), washed with water (2×10 mL), dried over Na₂SO₄, and evaporated. Thecrude residue was purified by normal phase chromatography with 0-10%MeOH—CH₂Cl₂ to give the desired target OS0032 (15 mg, 77%). ¹H-NMR (300MHz, CDCl₃): δ 8.85 (s, 8H), 8.43 (d, 6H, J=8), 8.18-8.33 (m, 10H),7.21-7.31 (m, 5H), 6.43 (m, 1H), 3.80-4.89 (m, 17H), 3.75 (d, 1H, J=7),3.19-3.34 (m, 9H), 2.97 (m, 6H), 2.31-2.42 (m, 4H), 1.97-2.18 (m, 3H),1.60-1.75 (M, 3h), 1.17-1.38 (M, 5H), 0.68-0.97 (m, 26H).

In FIG. 11 , a hydroxyamide intermediate (4) is formed from thecondensation of porphyrin carboxylate 2 with ethanolamine (3).

In one embodiment, the 3 is replaced by aminoalcohols L5 or L6, whereinthe group P represents H and n=1-12.

In another embodiment, MMAE (7) may be replaced with auristatin-relatedpeptides T5c, T9c and T10c (Table 8). MMAE is denoted as T4c in Table 8.

In another embodiment, porphyrin (2) may be replaced by P1, where thecarboxylic acid substituent (COOH) occupies ortho, meta or para, thatis, positions 2, 3 or 4, and wherein the three substituents L, M and Nmay independently occupy the ortho, meta or para (positions 2, 3 or 4)on their respective aromatic rings. The substituents L, M ad N areselected from the set consisting of:

-   -   a) H;    -   b) carboxyaryl esters and acids (COOR) where the R may be H,        lower straight chain or branched alkyl, cycloalkyl,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, hetero-substituted cycloalkyl and        additionally, sugars. In addition, R may include aryl and        heteroaryl substituents;    -   c) Carboxamides (CONR1R2), where wherein the R1 and R2 groups        may individually be H, be lower straight chain or branched        alkyl, cycloalkyl, hydroxy-substituted alkyl, polyethers (lower        PEG), amino-substituted alkyl, hetero-substituted cycloalkyl. In        addition, R may include aryl and heteroaryl substituents;    -   d) Aminoaryl groups (NR1R2) where R1 or R2 may individually be        selected from the group lower alkyl, branched lower alkyl,        cycloalkyl, or hetero-substituted alkyl or hetero-substituted        cycloalkyl. R may also be an acyl group such as C(O)R, where R        may include lower alkyl, hydroxy-substituted alkyl, polyethers        (lower PEG), amino-substituted alkyl, cycloalkyl. In addition, R        may include aryl and heteroaryl substituents;    -   e) Sulfur containing functional groups that may include thiols,        sulfonic acids and corresponding amides thereof. The amides may        be further defined as containing a nitrogen moiety NHR, with the        N connected by a single bond to the sulfur atom, and where R is        lower alkyl, cycloalkyl, or hetero-substituted alkyl or        hetero-substituted cycloalkyl. R may also be an acyl group such        as C(O)R, where R may include lower alkyl, hydroxy-substituted        alkyl, polyethers (lower PEG), amino-substituted alkyl,        cycloalkyl. In addition, R may include aryl and heteroaryl        substituents;    -   f) Lower straight chain or branched alkyl groups, cycloalkyl        groups, hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl;    -   g) Oxygen groups such as hydroxy and substituted hydroxyaryl (OH        or OR), where R may be chosen from the group including lower        straight chain or branched alkyl groups, cycloalkyl groups,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl. In addition, R may include acyl (C(O)R),        carbamoyloxy (C(O)NR1R2), where R1 and R2 may be H or lower        alkyl, aryl and heteroaryl substituents.

In a preferred embodiment, the aminoalcohol L5 is chosen such that P isH and n=1. The porphyrin in this embodiment is selected to be P1,wherein the carboxylic acid group is in the para position relative tothe porphyrin ring, L, M and N are all carboxylic acid or carboxymethylester groups and are all in the para position relative to the porphyrinring. Moreover, the toxin is MMAE (T4c).

The procedure for synthesis of target OS0025 is illustrated in FIG. 12and described below.

Preparation of1-(3-hydroxypropyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-ium(3). A mixture of 1 (1.26 g, 2.03 mmol) and 3-bromo-1-propanol 2 (0.43g, 2.36 mmol) in a mixture of EtOH and CHCl₃ (400 mL, 1:3) was refluxedfor 6 d. The solvents were evaporated and absorbed on silica forpurification. The silica absorbed crude compound was purified by twoshort silica gel columns (CH₂Cl₂:EtOH, 7:3, v/v) to get the desiredcompound 3 (74 mg, 6%). ¹H-NMR (300 MHz, DMSO-d₆): δ 11.9 (s, 1H), 9.5(d, 2H), 8.81-9.08 (m, 16H), 8.27 (d, 6H), 5.00-5.03 (m, 3H), 2.75-2.77(m, 2H), 2.32-2.35 (m, 2H). MS m/z=677 [M]⁺.

Preparation of1-(3-((((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)propyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-ium(OS0025). To a mixture of 3 (23 mg, 0.034 mmol) and 4 (13 mg, 0.05 mmol)in DMSO-d₆ (1 mL) was added DIPEA (9 μL, 0.05 mmol). The resultantmixture was stirred at ambient temperature overnight. The HPLC showednew peak and consumption of 3. At this point of time, was added 5 (20mg, 0.034 mmol) followed by DIPEA (18 μL, 0.1 mmol). The reactionmixture was stirred at room temperature for 7 h and stored at 0° C. over64 h. The mixture was diluted with 9 mL of chloroform. The supernatantlayer was collected after centrifugation (15 min, 3000 rpm) andevaporated. The residue was purified by reverse phase ISCO withwater-acetonitrile (0.1% TFA) to get the desired compound 6 (12.5 mg,30%) as purple solid. MS (ESI) m/z=1247 [M]⁺. Purity by HPLC(ELSD): >89%, t_(R)=3.58.

HPLC Condition.

Agilent Tech 1100 series HPLC System equipped with Variable WavelengthDetector and ELSD Detector and UV at 254 nm

Column: Agela, Durashell C18, 3.0 μm, 4.60×50 mm.

Mobile Phase: A (ACN with 0.1% TFA)Mobile Phase: D (H₂O with 0.1% TFA)

Gradient

Time A (ACN with 0.1% TFA) D (H₂O with 0.1% TFA) 0 5 95 5.75 95 5 8 95 59 5 95

The synthetic scheme illustrates the alkylation ofmeso-tetrakis(4-pyridyl)porphyrin (1) with 1-bromo-3-propanol (2) toafford pyridinium intermediate 3 via an S_(N)2 reaction. In anembodiment, the bromoalcohol (2) may be replaced with relatedbromoalcohols L7 and L8, where n is selected from 1-12.

The porphyrin 1 may also be substituted by P2, wherein the position ofthe heteroatom is one of X, Y or Z on the four aromatic rings. Thus, oneach pyridine ring of P2, a nitrogen (N) is positioned at either X or Yor Z, with CH occupying the remaining two positions. For example, theporphyrin meso-tetrakis(3-pyridyl)porphyrin could be represented as Y isN, X and Z are CH on all four pyridine rings. In a general embodiment,the position of the N on each ring may differ. In a more preferredembodiment, the position of the N atom in each pyridine ring is the samein all four pyridine rings. In a preferred embodiment, the porphyrinmeso-tetrakis(4-pyridyl)porphyrin could be represented as X=N, Y and Zare CH on all four pyridine rings.

FIG. 12 further illustrates the reaction of the activated carbonate 4with the aminosugar of doxorubicin to afford the carbamate moiety inOS0025. In another embodiment, doxorubicin may be replaced with ananthracycline selected from the group T1b-T4b, wherein T1b isdoxorubicin (Table 6).

In a preferred embodiment, P2 is selected such that X=N, Y=Z=CH; L7 isselected such that n=1; and Tnb is selected as T1b (doxorubicin).

The procedure for the synthesis of target OS0029 is illustrated in FIG.13 and described below.

Preparation of1-(3-hydroxypropyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-ium(3). A mixture of 1 (1.26 g, 2.03 mmol) and 2 (3.31 g, 23.86 mmol) in amixture of EtOH and CHCl₃ (400 mL, 1:3) was refluxed for 6 d. Thesolvents were evaporated and adsorbed on silica for purification. Thesilica adsorbed crude compound was purified by two successive shortsilica gel columns (CH₂Cl₂:EtOH, 7:3, v/v) to get the desired compound 3(74 mg, 6%). ¹H-NMR (300 MHz, DMSO-d₆): δ 11.9 (s, 1H), 9.5 (d, 2H),8.81-9.08 (m, 16H), 8.27 (d, 6H), 5.00-5.03 (m, 3H), 2.75-2.77 (m, 2H),2.32-2.35 (m, 2H). MS m/z=677 [M]⁺.

Preparation of(S)-1-(3-((4-(4-(4-amino-3-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)phenyl)-1H-pyrazol-1-yl)piperidine-1-carbonyl)oxy)propyl)-4-(10,15,20-tri(pyridin-4-yl)porphyrin-5-yl)pyridin-1-ium(OS0029). To a mixture of 3 (30 mg, 0.044 mmol) and disuccinimidylcarbonate (13.6 mg, 0.05 mmol) in DMSO-d₆ (1.5 mL) was added DIPEA (11μL, 0.066 mmol). The resultant mixture was stirred at ambienttemperature overnight. The HPLC showed new peak and consumption of 3. Atthis point of time, was added crizotinib hydrochloride (25.8 mg, 0.053mmol) followed by DIPEA (22 μL, 0.066 mmol). Then the mixture wasstirred at room temperature for 7 h and stored at 0° C. over 64 h. Thecrude mixture was loaded on reverse phase column and eluted with 0-100%acetonitrile-water (0.1% TFA). The pure fractions were collected andlyophilized. The solid was triturated/sonicated with chloroform (5 mL×4)and the supernatant layer was separated. The remaining solid was driedunder vacuum overnight to yield 6 (23 mg, 45%). MS (ESI) m/z=1152.3[M]⁺. Purity by HPLC (ELSD): >97%, t_(R)=4.58.

HPLC Condition.

Agilent Tech 1100 series HPLC System equipped with Variable WavelengthDetector and ELSD Detector

Column: Agela, Durashell C18, 3.0 μm, 4.60×50 mm.

Mobile Phase: A (ACN with 0.1% TFA)Mobile Phase: D (H₂O with 0.1% TFA)

Gradient

Time A (ACN with 0.1% TFA) D (H₂O with 0.1% TFA) 0 5 95 5.75 95 5 8 95 59 5 95

Detection: UV at 254 nm

FIG. 13 illustrates the alkylation of meso-tetrakis(4-pyridyl)porphyrin(1) with 1-bromo-3-propanol (2) to afford hydroxypyridinium intermediate3 via an S_(N)2 reaction. In an embodiment, the bromoalcohol (2) may bereplaced by L7 and L8, wherein n=1-12. The porphyrin 1 may be replacedwith porphyrins P2, wherein the position of the heteroatom is one of X,Y or Z on the four aromatic rings. Thus, on each pyridine ring of P2, anitrogen (N) is positioned at either X or Y or Z, with CH occupying theremaining two positions. For example, the porphyrinmeso-tetrakis(3-pyridyl)porphyrin could be represented as Y is N, X andZ are CH on all four pyridine rings. In a general embodiment, theposition of the N on each ring may differ. In a more preferredembodiment, the position of the N atom in each pyridine ring is the samein all four pyridine rings. In a preferred embodiment, the porphyrinmeso-tetrakis(4-pyridyl)porphyrin could be represented as X=N, Y and Zare CH on all four pyridine rings. FIG. 13 further illustrates thereaction of 3 with disuccimidyl carbonate to form an activated carbonateintermediate, which is subsequently reacted in Step 3 with the secondaryamine of crizotinib to afford OS0029.

Further, the crizotinib may be replaced in this condensation reaction bykinase inhibitors (T1a-T33a), as shown in Table 7.

In a preferred embodiment, P2 is selected such that X=N, Y=Z=CH; L7 isselected such that n=1; and crizotinib is the toxin.

The procedure for synthesis of target OS0023 is illustrated in FIG. 14and described below.

Synthesis of ethyl5-(3-(10,15,20-tris(3-hydroxyphenyl)porphyrin-5-yl)phenoxy) pentanoate)(2). A mixture of 1 (1.25 g, 1.84 mmol) and K₂CO₃ (0.5 g) in DMF (30 mL)was stirred under nitrogen at room temperature for 30 min. Ethyl5-bromopentanoate (1.15 g, 5.5 mmol, 3 eq.) was added. The mixture wasstirred at room temperature overnight. The mixture was diluted with DCM,washed with water, satd. NaHCO₃ (aq), water and brine, dried overNa₂SO₄, and evaporated. The crude residue was purified with twosuccessive column chromatography to give 2 (0.42 g, 28%). ¹H-NMR (300MHz, DMSO-d₆): δ 12.0 (s, 1H), 9.90 (s, 3H), 8.88 (s, 8H), 7.66-7.79 (m,3H), 7.52-7.65 (m, 9H), 7.36-7.7.42 (m, 1H), 7.18-7.26 (m, 3H),4.12-4.20 (m, 2H), 4.05 (q, 2H), 2.34-2.40 (m, 2H), 1.65-1.85 (m, 4H),1.12 (t, 3H).

Synthesis5-(3-(10,15,20-tris(3-hydroxyphenyl)porphyrin-5-yl)phenoxy)pentanoicacid (3). To a solution of 2 (157 mg, 0.19 mmol) in THF (45 mL) wasadded a solution of LiOH—H₂O (0.15 g) in water (30 mL) at roomtemperature. The mixture was stirred at room temperature overnight.Evaporated THF, diluted with satd. NH₄Cl (aq), extracted with DCM,washed with brine, and evaporated. The intermediate was used for nextstep reaction without further purification. 3 (0.14 g, 92%) ¹H-NMR (300MHz, DMSO-d₆): δ 12.0 (s, 2H), 9.90 (s, 3H), 8.89 (s, 8H), 7.66-7.82 (m,3H), 7.54-7.65 (m, 9H), 7.36-7.7.42 (m, 1H), 7.20-7.26 (m, 3H),4.14-4.22 (m, 2H), 2.26-2.36 (m, 2H), 1.65-1.85 (m, 4H).

Synthesis5-(3-(10,15,20-tris(3-acetoxyphenyl)porphyrin-5-yl)phenoxy)pentanoicacid (4). To a mixture of 3 (67 mg, 0.068 mmol) in 1% TfOH/CH₃CN (25 mL)was added AcCl (1.5 mL) at room temperature. The reaction was stirred atsame temperature for 2 h then poured into cold half concentrated NaHCO₃and EtOAc, extracted with EtOAc, washed with brine and solventsevaporated. The crude residue was purified with column chromatography togive 4 (75 mg, 95%) ¹H-NMR (300 MHz, CDCl₃): δ 12.1 (s, 1H), 8.91 (s,8H), 8.03-8.10 (m, 3H), 7.92-7.98 (m, 3H), 7.70=7.81 (m, 5H), 7.58-7.64(m, 1H), 7.49-7.56 (m, 3H), 7.32-7.28 (m, 1H), 4.05-4.20 (m, 2H),2.28-2.35 (m, 2H), 2.37 (s, 9H), 1.84-194 (m, 4H).

Synthesis(S)-(20-(3-((5-(4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-5-oxopentyl)oxy)phenyl)porphyrin-5,10,15-triyl)tris(benzene-3,1-diyl)triacetate (5). To a solution of 4 (80 mg, 0.087 mmol) in DMF (6 mL) wasadded HATU (77 mg, 0.2 mmol, 2.3 eq.) and DIPEA (104 μL. Stirred at roomtemperature for 5 min. crizotinib-HCl (47 mg, 0.096 mmol, 1.1 eq.) wasadded. The mixture was stirred at room temperature overnight. The DMFsolvent was evaporated, the residue was dissolved to EtOAc, then washedwith water and brine. The crude residue was purified by a normal phasecolumn chromatography to afford 5 (71 mg, 61%); MS [M+1]⁺=1336.

Synthesis(S)-1-(4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-5-(3-(10,15,20-tris(3-hydroxyphenyl)porphyrin-5-yl)phenoxy)pentan-1-one(OS0023). To a solution of 5 (71 mg, 0.053 mmol) in MeOH/DMF (20 mL/6mL) was added a solution of NaOMe in methanol (3.2 mL, 50 mM, 1 eq.) at0° C. The reaction was monitored by HPLC. Stirred at same temperaturefor 1 h then warmed to room temperature. After stirring at roomtemperature for 2 h, HPLC indicated that the reaction was complete. Thereaction was quenched with a solution of HOAc in MeOH (0.4 mL/2 mL).Concentrated, the residue was dissolved into EtOAc, washed with waterand brine. After evaporating the solvents, the residue was purified by anormal phase column chromatography to afford the desired target OS0023(35 mg, 55%). MS [M+1]+=1210.

FIG. 14 illustrates the condensation of the secondary amine ofcrizotinib with the carboxylate group of intermediate 4 using thepeptide coupling reagent HATU to afford intermediate 5. In an embodimentcrizotinib may be replaced with other kinase inhibitors chosen from theset illustrated in Table 7, wherein crizotinib is compound 1 in Table 7.

Moreover, in another embodiment, the ethyl 5-bromopentanoate used in thesynthesis of OS0023 may be replaced with either L1 or L2, wherein Yrepresents OH and the n is selected from 1-12 for L1 and L2. Xrepresents a leaving group suitable for an S_(N)2 reaction with anucleophile, such as a phenol or its conjugate base. In this context,the leaving group X is either a halogen leaving groups (Cl, Br, I) or avariety of activated sulfonyl esters such as mesylates, tosylates ortriflates.

FIG. 14 illustrates the alkylation ofmeso-tetrakis(3-hydroxyphenyl)porphyrin (1) to afford intermediate 2. Inyet another embodiment, the phenolic porphyrin 1 is replaced byporphyrin P4, wherein the hydroxy substituent could be situated at theortho, meta or para positions (positions 2, 3 or 4) of the aromatic ringand, moreover, where the substituents L, M and N, may be independentlyoccupy the ortho, meta or para, that is 2-, 3- or 4-positions, on theirrespective aromatic rings. The substituents L, M ad N are selected fromthe group consisting of:

-   -   a) H;    -   b) carboxyaryl esters and acids (COOR) where the R may be H,        lower straight chain or branched alkyl, cycloalkyl,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, heterosubstituted cycloalkyl and        additionally, sugars. In addition, R may include aryl and        heteroaryl substituents;    -   c) Carboxamides (CONR1R2), where wherein the R1 and R2 groups        may individually be H, be lower straight chain or branched        alkyl, cycloalkyl, hydroxy-substituted alkyl, polyethers (lower        PEG), amino-substituted alkyl, heterosubstituted cycloalkyl. In        addition, R may include acyl (C(O)R1), carbamoyloxy (C(O)NR1R2),        aryl and heteroaryl substituents;    -   d) Aminoaryl groups (NR1R2) where R1 or R2 may individually be        selected from the group lower alkyl, branched lower alkyl,        cycloalkyl, or heterosubstituted alkyl or heterosubstituted        cycloalkyl. R may also be an acyl group such as C(O)R, where R        may include lower alkyl, hydroxy-substituted alkyl, polyethers        (lower PEG), amino-substituted alkyl, cycloalkyl. In addition, R        may include aryl and heteroaryl substituents;    -   e) Sulfur containing functional groups that may include thiols,        sulfonic acids and corresponding amides thereof. The amides may        be further defined as containing a nitrogen moiety NHR, with the        N connected by a single bond to the sulfur atom, and where R is        lower alkyl, cycloalkyl, or heterosubstituted alkyl or        heterosubstituted cycloalkyl. R may also be an acyl group such        as C(O)R, where R may include lower alkyl, hydroxy-substituted        alkyl, polyethers (lower PEG), amino-substituted alkyl,        cycloalkyl. In addition, R may include aryl and heteroaryl        substituents.    -   f) Lower straight chain or branched alkyl groups, cycloalkyl        groups, hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and heterosubstituted        cycloalkyl;    -   g) Oxygen groups such as hydroxy and substituted hydroxyaryl (OH        or OR), where R may be chosen from the group including lower        straight chain or branched alkyl groups, cycloalkyl groups,        hydroxy-substituted alkyl, polyethers (lower PEG),        amino-substituted alkyl, cycloalkyl and hetero-substituted        cycloalkyl. In addition, R may include acyl, aryl and heteroaryl        substituents; and    -   h) Cyano or halogens (F, Cl, Br, I).

In a preferred embodiment utilizing L1, n=4, X is bromo and Y is ethoxy.Moreover, P4 is selected such that L, M and N are all hydroxyl in thepara position relative to the porphyrin ring. The toxin is selected tobe crizotinib.

The cytotoxic activity and/or efficacy of a PAC compound is evaluated asdescribed below. A set of tumor and normal cell lines are evaluated invitro. The CellTiter-Glo® Assay (Promega Corporation) is used todetermine the cytotoxicity activity of an individual PAC compound asdisclosed herein over a range of concentrations to determine adose-response curve. The PAC compounds to be tested are kept in thedark, at −20° C., until use. Abbreviations:TCPP=tetra(4-carboxyphenyl)porphyrin;THPP=meso-tetrakis(3-hydroxyphenyl)porphyrin. Exemplary cell linesselected for the assay are listed in Table 10. Entries 1-4 are cancercell lines while entries 5-6 are normal cell lines.

TABLE 10 Cell lines for in vitro assay Entry Cell Line Tissue NCI-60line 1 NCI-H460 Lung yes 2 MDA-MB-231 Breast (triple negative) yes 3HepG2 Liver no 4 PC3 Prostate yes 5 Lonza CC-2547 SAEC-Small AirwayEpithelial no Cells 6 Lonza CC-2551 HMEC-Mammary Epithelial no Cells

Cell culture conditions: cells are thawed, grown and split twice priorto conducting the cytotoxicity assay, or until satisfactory cell growthhas been established. Cells are grown in their appropriate medium; theculture media for cancer cell lines will be supplemented with 5% fetalbovine serum (not heat-inactivated) and anti-bacterial and/or fungalagents. For the cultures of normal cell lines, the culture mediumspecified by the vendor will be used. For the CellTiter-Glo® Assay,cells are plated in 96-well plates with one cell line per plate. On eachplate, the Assay is conducted on cells exposed to a selected PACcompound as disclosed herein (8 dose levels, 3 replicates at each doselevel). On the same plate, cells are exposed to the unreacted cytotoxicagent that corresponds to the cytotoxic agent incorporated into the PACcompound being tested on the plate as a positive control (3 replicates).On each plate, the Assay is conducted on cells grown in theirappropriate medium, without any of the above-mentioned additives (3replicates). This is the negative control of the experiment.

The cell assay is ideally conducted in the dark or low light conditions.A PAC compound is dissolved in a 100% DMSO solution and tested in theassay over a concentration range as shown in Table 11.

TABLE 11 Dose range for testing PAC compounds and cytotoxins. DoseConcentrations (nM) 1 2 3 4 5 6 7 8 10,000 3,165 1,001 317 100 32 10.03.2For example, a positive control such as Doxorubicin is used at 10 uMdoxorubicin.

Media (negative control): Since the PAC compounds and cytotoxic agentsare dissolved in 100% DMSO, the negative control of “medium only” isDMSO as well. The percentage of DMSO in the “medium only” control isequal that of the highest DMSO concentration in the concentration rangeof PAC compounds and cytotoxins.

CellTiter-Glo® Assay should is performed 72 hours after first exposingcells to the various PAC compounds, to discriminate betweenmetabolically active (indicating live, quiescent, and senescent cells)and metabolically inactive (dead) cells.

In Vivo Testing of PAC compounds

In order to evaluate the efficacy of a particular PAC compound for aparticular medicinal application, the compounds are first tested againstappropriately chosen test cells in vitro. In a non-limiting example, PACcompounds are tested against tumor cells, for example, lung tumor cellsin murine in vivo models.

Animals: Male nude (nu/nu) mice 5-6 weeks of age weighing approximately22-25 g at the time of tumor implantation are used. Xenografts: Micewere implanted subcutaneously in the axilla region by trocar withfragments of NCI-H460 human non-small cell lung cancer tumors harvestedfrom s.c. growing tumors in nude mice hosts. When the tumors areapproximately 248-270 mm³ in size (11-15 days following implantation),the animals are pair-matched into treatment and control groups. Eachgroup contains 8 mice bearing tumors, each of which is ear-tagged andfollowed individually throughout the experiment.

Test Article Formulation Preparation: On each day of dosing, theporphyrin conjugate is weighed and the appropriate volume of DMSO addedfor initial dissolution. To this is added either 5% dextrose in water(D5W), isotonic saline or glycerol, or a combination of the above, forpreparation of the stock solution. Thus, a stock solution, 0.1-1.0 mg/mLis then diluted to lower concentrations through serial dilutions. Dosingsolutions are prepared immediately prior to dosing in sterilescintillation vials. Similarly, the parent porphyrin and toxin controlcompounds are formulated as per the conjugate.

Compound Dose Preparation: Preparation process is developed based oncompound properties in vehicles suitable for parenteral administrationto the test animals. Conjugates are dissolved in DMSO and then dilutedwith one or a combination of the following diluents: isotonic saline,D5W or glycerol to prepare stock solutions, which are further seriallydiluted for intraperitoneal (IP) or intravenous (i.v.) administration.The dose ranges for the conjugate, parent porphyrin and parent toxin are1-100 mg/kg.

Dosing Solution Storage: Prepared test article dosing solutions used onthe day of preparation are maintained at controlled ambient temperaturein the absence of light and during dosing and sampling. Prepared testarticle dosing solutions not used on the day of preparation arediscarded.

Dosing Procedure: The administration of vehicle or test agents beginsthe same day as pair-matching (Day 11). The doses are administered by IPor i.v. injection at a constant dose volume of 10 mL/kg based upon eachanimal's body weight at that time.

Tumor Volume: Tumor volumes are monitored twice weekly by measuring thewidth (mm) and length (mm) of the tumor mass using digital calipers.Tumor measurements are converted to a tumor volume (mm³) using theformula, {width (mm)²×length (mm)}×0.52.

Body Weight: All mice are individually weighed prior to each dose, butonly recorded twice weekly. Clinical Observations: Abnormal clinicalsigns are recorded for all mice before each dosing and frequently aftereach dose. Abnormal clinical signs are recorded on all mice at the timeof body weight measurements on non-dosing days. Mortality evaluationsare performed on all mice daily.

Data Analysis: In this experiment the tumor growth inhibition and tumorgrowth delay for each treatment group compared to their respectivecontrol group is reported. Tumor growth inhibition (T/C) is calculatedusing the mean tumor volume from each group on the day the mediancontrol mouse volume reaches 1000 mm³. Tumor growth delay utilized thetime required for the median mouse in each group to reach the same tumorvolume endpoint of 2000 mm³. This data is reported as T-C and T/C tumorgrowth delay. The classification of antitumor activity of each treatmentgroup is based on similar parameters found in two publications (Hill,2001; Johnson, et al., 2001), the latter reference from the NationalCancer Institute (Bethesda, MD). The table below summarizes thesefinding.

Rating Tumor Growth Inhibition Tumor Growth Delay (T/C) Active<60% >1.5x  Moderate <40% 1.75x High <10% 2x  

Partial and complete regressions are also monitored. A partialregression occurs when a tumor regresses by 50% or more compared to itssize at the time of first dosing. A tumor is notated as a completeregression when the tumor is no longer visible or palpable. Toxic deathsare deaths that occur during the course of dosing or immediatelyfollowing the conclusion of dosing. For recording of the percent bodyweight change in each group, the following formula was employed: (Day‘x’ mean weight−Day 1 mean weight)/Day 1 weight×100%. The % maximalweight loss for each group was the maximum weight loss which occurredduring the first two weeks following drug administration.

Note that in the specification and claims, “about” or “approximately”means within +/−twenty percent (20%) or in a preferred embodiment+/−tenpercent (10%) of the numerical amount cited. Although the invention hasbeen described in detail with particular reference to these preferredembodiments, other embodiments can achieve the same results. Forexample, the porphyrin cytotoxic conjugate can be combined with abiodegradable matrix material (such as alginate, polylactic acid,polyglycolic acid, caprolactone etc) and implanted in a tissue such thatthe porphyrin cytotoxic agent is delivered to the tissue over time asthe biodegradable matrix dissolves. Variations and modifications of thepresent invention will be obvious to those skilled in the art and it isintended to cover all such modifications and equivalents. For example,in the formula Pn-Ln-Tn, the n of Ln may be selected from 1-300 in oneembodiment of the present invention. The entire disclosures of allreferences, applications, patents, and publications cited above and/orin the attachments, and of the corresponding application(s), are herebyincorporated by reference.

1-47. (canceled)
 48. A composition comprising: a compound of Formula III

or a pharmaceutically acceptable salt thereof, wherein an A₁, A2, A3 andA4 are each covalently attached to a porphyrin ring of Formula III andA₁, A2, A3, and A4 are independently selected from a substitutedaromatic ring or a six membered heteroaromatic ring containing a singlenitrogen atom at the 2, 3 or 4 position relative to the porphyrin ring;B₁ is a covalent linker moiety which connects A₁ to a cytotoxic agent Z₁and is selected from

wherein n is selected from 1-12; and the Z₁ is a cytotoxic agentselected from


49. The composition of claim 48 or the pharmaceutically acceptable saltthereof, further including a pharmaceutical acceptable carrier.
 50. Thecomposition of claim 49 or the pharmaceutically acceptable salt thereof,wherein the pharmaceutically acceptable carrier is a liquid carrierselected from saline, glucose, alcohols, glycols, esters, amides, andany combination thereof.
 51. The composition of claim 48 or thepharmaceutically acceptable salt thereof, wherein the compound is in adosage form and the dosage form is parenteral and the dosage form isselected from intradermal dosage form, a subcutaneous dosage form, anintramuscular dosage form, a subcutaneous dosage form, an intravenousdosage form, an intrathecal dosage form, and an epidural dosage form.52. The composition of claim 48 or the pharmaceutically acceptable saltthereof, wherein the compound is in a dosage form and the dosage form isnonparenteral and the dosage form is selected from oral dosage form,sublingual dosage form, topical dosage form, transdermal dosage form,ophthalmic dosage form, otic dosage form, nasal dosage form, rectaldosage form, and vaginal dosage form.
 53. The composition of claim 48 orthe pharmaceutically acceptable salt thereof, wherein the substitutedaromatic ring of the A₁ comprises a carboxylic amide functional group ateither an ortho, meta, or para position with respect to the porphyrinring and wherein A2, A3 and A4 are each a substituted aromatic ringwherein each A2, A3, and A4 substituted aromatic ring has a substituentat either a ortho, meta or para position with respect to the porphyrinring and the substituent is either a carboxylic acid or carboxylicmethyl ester.
 54. The composition of claim 48 or the pharmaceuticallyacceptable salt thereof, wherein the substituted aromatic ring of theA2, A3, and A4 comprises a carboxylic methyl ester in a para positionwith respect to the porphyrin ring and the carboxylic amide of A1 is inthe para position with respect to the porphyrin ring.
 55. Thecomposition of claim 48 or the pharmaceutically acceptable salt thereof,wherein B₁ is L11 or L13.
 56. The composition of claim 48 or thepharmaceutically acceptable salt thereof, wherein Z₁ is selected fromT1b, 1 and T4c.
 57. The composition of claim 48 or the pharmaceuticallyacceptable salt thereof, wherein the substituted aromatic ring of A₁comprises an aromatic ether functional group at either an ortho, meta orpara position with respect to the porphyrin ring, and wherein A2, A3 andA4 are each the substituted aromatic ring wherein each A2, A3, and A4substituted aromatic ring has a substituent located at an ortho, meta orpara position with respect to the porphyrin ring wherein the substituenton each A2, A3, and A4 substituted aromatic ring is independentlyselected from lower alkyl, branched lower alkyl, cycloalkyl, halogens(F, Cl, Br, I), cyano, amino or substituted amino, sulfonic acid orsulfonamide, aromatic ether, aromatic hydroxyl, carboxylic acid alkylesters or carboxylic acid amide.
 58. The composition of claim 48 or thepharmaceutically acceptable salt thereof, wherein B₁ is selected fromL9, L10, L15, and L16.
 59. The composition of claim 58 or thepharmaceutically acceptable salt thereof, wherein a substituent of thesubstituted aromatic ring at position A2, A3 and A4 is a hydroxyl andmay occupy the ortho, meta or para position with respect to theporphyrin ring and B₁ is L9 or L15.
 60. The composition of claim 48 orthe pharmaceutically acceptable salt thereof, wherein the substitutedaromatic ring A₁ comprises an aromatic ether functional group, where theposition of the aromatic ether is meta with respect to the porphyrinring, and wherein A2, A3 and A4 are each the substituted aromatic ringwherein the substituent on the substituted aromatic ring is an aromatichydroxyl in the meta position with respect to the porphyrin ring, B₁ isL9 or L15 and Z₁ is selected from T1b, 1 and T4c.
 61. The composition ofclaim 48 or the pharmaceutically acceptable salt thereof, wherein thesix membered heteroaromatic ring of A₁ comprises a nitrogen atom where aposition of the nitrogen atom on the six membered heteroaromatic ringmay occupy one of a 2, 3 or 4 position with respect to the porphyrinring, A2, A3 and A4 are each a pyridine ring where the position of apyridine nitrogen on each pyridine ring of A2, A3 and A4 mayindependently occupy one of the 2, 3 or 4 position with respect to theporphyrin ring.
 62. The composition of claim 61 or the pharmaceuticallyacceptable salt thereof, wherein, B₁ is selected from L9, L10, L15, andL16.
 63. The composition of claim 61 or the pharmaceutically acceptablesalt thereof, wherein the six membered heteroaromatic ring comprisingthe nitrogen atom at A₁ is a pyridinium where the position of thenitrogen is in the 4 position with respect to the porphyrin ring, B₁ isL9 or L15, and Z₁ is selected from T1b, 1 or T4c.
 64. The composition ofclaim 63 or the pharmaceutically acceptable salt thereof, wherein the B₁is L9.
 65. The composition of claim 53 or the pharmaceuticallyacceptable salt thereof, wherein the compound is selected from


66. The composition of claim 57 or the pharmaceutically acceptable saltthereof, wherein the compound is selected from OS0023 and OS0024. 67.The composition of claim 61 or the pharmaceutically acceptable saltthereof, wherein the compound is selected from


68. The composition of claim 48 or the pharmaceutically acceptable saltthereof, for use in the treatment of cancer.
 69. The composition ofclaim 48 or the pharmaceutically acceptable salt thereof, for use in thetreatment of cancer cells in vitro.
 70. The composition of claim 48further comprising a cytotoxic agent wherein the cytotoxic agent is aformula that is the same or different than Z₁ of the compound of claim1.
 71. The composition of claim 63 wherein the different formula of thecytotoxic agent is selected from a class that is different as comparedto Z₁ of the compound of claim
 1. 72. A drug delivery device comprisingthe compound of Formula III of claim 48 enmeshed with a biodegradablepolymer.
 73. The drug delivery device of claim 72 wherein thebiodegradable polymer is selected from poly lactic co-glycolic acid,alginate, and polycaprolactone.
 74. The drug delivery device of claim 72wherein the compound is released over time when the drug delivery deviceis implanted into a patient.
 75. A kit comprising a compound of FormulaIII of claim 48 or a pharmaceutically acceptable salt thereof, and oneor more pharmaceutically acceptable carriers.
 76. A method of treatingcancer in a patient in need thereof comprising the steps of:administering to a patient in need thereof a therapeutically effectiveamount of a compound of Formula III of claim 48 or a pharmaceuticallyacceptable salt thereof.
 77. The method of claim 76 wherein thepharmaceutically acceptable carrier is a liquid carrier selected fromsaline, glucose, alcohols, glycols, esters, amides, and any combinationthereof.
 78. The method of claim 76 wherein the compound is in a dosageform and the dosage form is parenteral and the dosage form is selectedfrom intradermal dosage form, a subcutaneous dosage form, anintramuscular dosage form, a subcutaneous dosage form, an intravenousdosage form, an intrathecal dosage form, and an epidural dosage form.79. The method of claim 76 wherein the compound is in a dosage form andthe dosage form is nonparenteral and the dosage form is selected fromoral dosage form, sublingual dosage form, topical dosage form,transdermal dosage form, ophthalmic dosage form, otic dosage form, nasaldosage form, rectal dosage form, and vaginal dosage form.
 80. The methodof claim 76 wherein the substituted aromatic ring of the A₁ of thecompound comprises a carboxylic amide functional group at either anortho, meta, or para position with respect to the porphyrin ring andwherein A2, A3 and A4 are each a substituted aromatic ring wherein eachA2, A3, and A4 substituted aromatic ring has a substituent at either aortho, meta or para position with respect to the porphyrin ring and thesubstituent is either a carboxylic acid or carboxylic methyl ester. 81.The method of claim 80 wherein the substituted aromatic ring of the A2,A3, and A4 of the compound comprises a carboxylic methyl ester in a paraposition with respect to the porphyrin ring and the carboxylic amide ofA₁ is in the para position with respect to the porphyrin ring.
 82. Themethod of claim 76 wherein the B₁ of the compound is L11 or L13.
 83. Themethod of claim 76 wherein Z₁ of the compound is selected from T1b, 1 orT4c.
 84. The method of claim 76 wherein A2, A3 and A4 of the compoundrepresent substituted aromatic rings wherein the substituent is asulfonic acid or sulfonamide and may occupy the ortho, meta or paraposition with respect to the porphyrin ring.
 85. The method of claim 76wherein the substituted aromatic ring of A₁ of the compound comprises anaromatic ether functional group at either an ortho, meta or paraposition with respect to the porphyrin ring, and wherein A2, A3 and A4of the compound are each the substituted aromatic ring wherein each A2,A3, and A4 substituted aromatic ring has a substituent located at anortho, meta or para position with respect to the porphyrin ring whereinthe substituent on each A2, A3, and A4 substituted aromatic ring isindependently selected from lower alkyl, branched lower alkyl,cycloalkyl, halogens (F, Cl, Br, I), cyano, hydroxyl, amino orsubstituted amino, sulfonic acid or sulfonamide, aromatic ether,aromatic hydroxyl, carboxylic acid alkyl esters or carboxylic acidamide.
 86. The method of claim 76 wherein B₁ of the compound may beindependently selected from L9, L10, L15, L16.
 87. The method of claim79 wherein the substituent of the substituted aromatic ring at positionA2, A3 and A4 of the compound is a hydroxyl and may occupy either theortho, meta or para position with respect to the porphyrin ring and B₁is L9 or L15.
 88. The method of claim 76 wherein the substitutedaromatic ring A₁ of the compound comprises an aromatic ether functionalgroup, where the position of the aromatic ether is meta with respect tothe porphyrin ring, and wherein A2, A3 and A4 are each the substitutedaromatic ring wherein the substituent on the substituted aromatic ringis an aromatic hydroxyl in the meta position with respect to theporphyrin ring, B₁ is L9 or L15 and Z₁ is selected from T1b, 1 and T4c.89. The method of claim 76 wherein the six membered heteroaromatic ringof A₁ of the compound comprises a nitrogen atom where a position of thenitrogen atom on the six membered heteroaromatic ring may occupy one ofa 2, 3 or 4 position with respect to the porphyrin ring, A2, A3 and A4of the compound are each a pyridine ring where the position of apyridine nitrogen on each pyridine ring of A2, A3 and A4 mayindependently occupy one of the 2, 3 or 4 position with respect to theporphyrin ring.
 90. The method of claim 82 wherein the six memberedheteroaromatic ring comprising the nitrogen atom at A₁ is a pyridiniumwhere the position of the nitrogen is in the 4 position with respect tothe porphyrin ring, B₁ is L9 or L15, and Z₁ is selected from T1b, 1 orT4c.
 91. The method of claim 80 wherein the compound is selected from:


92. The method of claim 85 wherein the compound is selected from OS0023and OS0024.
 93. The method of claim 82 wherein the compound is selectedfrom